Methods and compositions for the selection and optimization of oligonucleotide tag sequences

ABSTRACT

Methods for selecting tag-oligonucleotide sequences for use in an in vitro nucleic acid assay. The selected tag sequences are useful for nucleic acid assay wherein interference between the nucleic acid sequences is the assay is to be controlled. Selected tag sequences are incorporated into nucleic acid assay to improve the performance of and/or minimize any interference between nucleic acids in the assay compared to untagged assays.

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims priority from U.S. provisional application Ser. No. 61/451,285 filed Mar. 10, 2011, incorporated herein by reference.

FIELD OF THE INVENTION

This present disclosure is directed to the field of nucleic acid-based assays incorporating one or more tag sequences into nucleic acid(s) of the assay. More specifically, methods and compositions are described related to the selection and optimization of oligonucleotide sequences referred to as “tags” for minimizing undesired nucleic acid interactions within the assay.

BACKGROUND

The use of short, user-selected (i.e., not defined by the target nucleic acid(s)) nucleic acid sequences (also known as “tags”) is a very powerful technique for the design and development of novel nucleic acid assay formats. These nucleic acid formats include nucleic acid amplification, sequencing or other assay formats.

Nucleic acid amplification provides a means for making more copies of a nucleic acid sequence that is relatively rare or unknown, for identifying the source of nucleic acids, or for making sufficient nucleic acid to provide a readily detectable amount. Amplification is useful in many applications, for example, in research, diagnostics, drug development, forensic investigations, environmental analysis, and food testing.

Typically, nucleic acid amplification uses one or more nucleic acid polymerase and/or transcriptase enzymes to produce at least one copy, and preferably multiple copies of a target nucleic acid sequence and, optionally, a tag sequence.

Many methods for amplifying nucleic acid sequences in vitro are known, including polymerase chain reaction (PCR), ligase chain reaction (LCR), replicase-mediated amplification, strand-displacement amplification (SDA), “rolling circle” types of amplification, and various Transcription Mediated Amplification (TMA) and reverse TMA (rTMA) methods. These known methods use different techniques to make amplified sequences, which usually are detected by using a variety of methods. See, for example, Schweitzer and Kingsmore, combining nucleic acid amplification and detection, current opinion in Biotechnology, 2001, 12, 21-27. These methods can be exemplified by the following publications (each of which is hereby expressly incorporated by reference): PCR—U.S. Pat. Nos. 4,683,195, 4,683,202, and 4,800,159; LCR—U.S. Pat. No. 5,516,663 and EP 0320308 B1; Replicase-mediated amplification—U.S. Pat. No. 4,786,600; SDA—U.S. Pat. No. 5,422,252A and U.S. Pat. No. 5,547,861; Rolling circle types of amplification—U.S. Pat. No. 5,714,320 and U.S. Pat. No. 5,834,252; TMA—U.S. Pat. Nos. 4,868,105, 5,124,246, 5,130,238, 5,399,491, 5,554,516 and 5,437,990, 5,824,518, US 2006-0046265 A1 and WO 1988010315 A1; rTMA—US 2006-0046265.

Amplification methods may introduce nucleic acid sequences into the sequence being amplified. Some methods use modified primers to introduce non-target nucleic acid sequences to the sequence being amplified. One example of a modified primer is a “tag” primer. A tag primer contains two parts: (1) a “tag sequence” that is a nucleic acid sequence that does not hybridize to the target nucleic acid sequence and (2) a primer sequence that is a nucleic acid sequence that does hybridize to the target nucleic acid sequence. The tag sequence is located 5′ to the primer sequence. The first round of amplification incorporates the tag sequence into the sequence being amplified. The second round of amplification uses primers that are complementary to the tag sequence.

Anchored PCR is a modified PCR method that uses an “adaptor” primer to amplify a sequence which is only partially known. See, for example, Loh et al., 1989, Science 243(4888):217-20; Lin et al., 1990, Mol. Cell. Biol. 10(4):1818-21).

Nested PCR may use primer(s) that contain a tag sequence unrelated to the target nucleic acid's target sequence to amplify nucleic acid from unknown target sequences in a reaction. See, for example, Sullivan et al, 1991, Electrophoresis 12(1):17-21; Sugimoto et al., 1991, Agric. Biol. Chem. 55(11):2687-92.

Other forms of amplification use a probe or probe set to introduce non-target priming sites located upstream and downstream of a target-specific sequence and adapter sequence(s). See, for example, U.S. Pat. Nos. 6,812,005 and 6,890,741, Fan et al. The two probes that bind in close proximity on a target sequence may be ligated together before being amplified by using the upstream and downstream universal priming sites.

Alternative assay methods may use probe hybridization and linear signal amplification by using a common sequence that is included in a variety of target nucleic acid-specific probes (e.g., US 20070111200, Hudson et al.). This method uses a labeled cassette that contains a sequence complementary to the common sequence to detect multiple target nucleic acids.

One problem that has heretofore existed with the use of tags is the lack of a rigorous selection method to identify the best tag for a given application. This has resulted in the use of less than optimal tags for particular applications.

Another problem is that tags may engage in undesired cross-reactions with other tags, primers, promoter providers, probes, amplicons, other target and non-target sequences and other such sequences in a given assay.

Another problem is that tags may engage in undesired cross-reaction with sequences in test samples from known or unknown sources, such as pathogenic or non-pathogenic organisms, mammalian nucleic acids, contaminating nucleic acids from enzymes, side-products of nucleic acid amplification reactions, etc.

One of the unsolved problems with multiplexed detection or amplification is the interference observed during multiplexing experiments, which limits the dynamic range, precision, and quantification characteristics for the target nucleic acids when present together in a sample.

In multiplex amplification reactions, a variety of undesired “side reactions” can occur that ultimately degrade assay performance. For instance, in the Transcription-Mediated Amplification (TMA) context, primers and/or promoter based amplification oligomers directed towards one target nucleic acid (or group of target nucleic acids) can interact with primers and/or promoter based amplification oligomers directed towards another target nucleic acid (or group of target nucleic acids), causing degraded performance of one, or the other or both amplification systems. This problem typically gets worse as the number of amplification systems present in a multiplex reaction increases. Other interactions that reduce assay performance include amplification oligomers interacting with one another or with other oligonucleotides in the amplification reaction, such as probes, blockers and target capture oligomers (TCOs), and amplicons. This problem of negative interaction between nucleic acids in a system can be reduced or solved by either converting all of the target specific primers/promoter providers in the assay or a portion of the target specific primers and/or promoter providers in the assay into primers and/or promoter based amplification oligomers containing tag sequences. Early rounds of amplification take place using these tagged amplification oligomers, thereby incorporating the tag sequences and their complements into the early amplification products. Subsequent rounds of amplification can take place using primers and/or promoter-based amplification oligomers having target hybridizing sequences directed to the incorporated tag sequence. In this way, the make-up of the subsequent round amplification oligomers is controlled by the user and undesired side reactions are reduced or eliminated. It is notable that the tag sequences used in two or more separate amplification oligomers can be the same sequence or different sequences.

Another related problem is the lack of a convenient procedure to quantitatively or qualitatively measure the relative levels of interferences in a multiplexed reaction.

Additionally, competition for amplification reaction resources may occur in multiplex amplification reactions when the same tag sequence is used for multiple tagged amplification oligomers. For example, in multiplex amplification reactions, target nucleic acid present at high levels will consume the amplification oligomers complementary to the tag sequence much faster than target nucleic acid present at low levels. The inability to uniformly amplify the target nucleic acids due to amplification resource competition may lead to false negatives because the target nucleic acids present at low levels are not amplified to a detectable amount.

One possible solution to these problems is to create unique tag sequences for incorporation into one or more oligomers in an assay. The unique tags are designed such that they do not interact with each other, or with any other sequences in the assay. In this way, an assay incorporating one or more tag sequences can proceed without reduced performance caused by the undesired interaction of various nucleic acids, including tag sequences, present in the assay reaction.

Clearly, there are numerous problems in the art of using nucleic acid tag sequences in a nucleic acid assay. It would be desirable to have tag sequences and methods for identifying tag sequences that are useful in a nucleic acid assay and that avoid the problems. It would be desirable to have methods for rapidly identifying tag sequences for use in a nucleic acid assay.

SUMMARY OF THE INVENTION

It is, therefore, our object of the present invention to provide an identification and selection method that can be used to generate unique tag sequence sets. Thus, the invention encompasses methods to identify and select nucleic acid tags for use in nucleic acid assays, sequencing, amplification, manipulation, interaction and other processing (sometimes referred to herein genetically as “nucleic acid assays”). The invention also encompasses a method of minimizing interference between nucleic acid sequences present in an assay, including amplification assays, multiplex amplification assays, sequencing assays and the like. In addition, the invention also encompasses compositions which have been selected by these methods.

The present invention encompasses a method for identifying a nucleic acid tag sequence for use in a nucleic acid assay, comprising: a) generating a pool of nucleic acid sequences, wherein the pool is at least three nucleic acid sequences; b) screening the pool of nucleic acid sequences to identify two or more nucleic acid sequences have two or more performance characteristics the; and c) selecting one or more nucleic acid sequences, each for use as tag sequence in a nucleic acid assay.

The invention further includes a method as described above, further comprising: d) comparing a nucleic acid sequences from the pool of nucleic acid sequences against a database having one or more nucleic acid sequences to determine complementarity of the nucleic acid sequences from the pool of nucleic acid sequences to the database having one or more sequences; e) generating a sub-pool of nucleic acid sequences, wherein the sub-pool is a collection of nucleic acid sequences with complementarity that is less than 95% to the nucleic acid sequence(s) in the database, that is less than 90% to the nucleic acid sequence(s) in the database; that is less than 80% to the nucleic acid sequence(s) in the database, that is less than 70% to the nucleic acid sequence(s) in the database, or that is less than 50% to the nucleic acid sequence(s) in the database; f) screening the sub-pool of nucleic acid sequences for one or more performance characteristics selected from melting temperature, activity in an enzyme reaction, G-C content, hybridization energy, multimer formation, internal structure formation, G-quartet formation, and hairpin-stability; and g) selecting one or more nucleic acid sequences from the sub-pool for use as tag sequences in a nucleic acid assay.

The invention further includes a method as described above, further comprising: h) synthesizing at least two different oligonucleotides for use in a nucleic acid assay, wherein each of the synthesized oligonucleotides has a tag sequence selected according to step g); and i) measuring for each of the different oligonucleotides synthesized in step h) one or more of the following performance characteristics: speed of reaction, limit of detection, interference, precision of replicates, performance against a specific target nucleic acid sequence, or performance against multiple target nucleic acid sequences in a nucleic acid assay, and optionally comparing the measurements to the measurements obtained for an untagged oligonucleotide; and j) selecting one or more of the nucleic acid tag sequences used in step i) for use in a nucleic acid assay.

The invention further includes a method as described above, further comprising the steps of: k) modifying the sequence of the tag sequence incorporated into an oligonucleotide from step h) to obtain a modified tag sequence for incorporation into an oligonucleotide; 1) measuring for the oligonucleotide containing a modified tag sequence from step k) one or more of the following performance characteristics: speed of reaction, limit of detection, interference, precision of replicates, performance against a specific target nucleic acid sequence, or performance against multiple target nucleic acid sequences in a nucleic acid assay; and m) selecting one or more of the modified nucleic acid tag sequences used in step i) for use in a nucleic acid assay.

The invention further includes a method as described above, wherein the modification in step k) comprises systematically deleting nucleotides from the tag sequence.

The invention further includes a method as described above, further comprising at step g), the steps of: (i) modifying the sequence of the tag sequence from step g); (ii) synthesizing an oligonucleotide to contain the modified tag sequence; (iii) measuring for the oligonucleotide containing a modified tag sequence one or more of the following performance characteristics: speed of reaction, limit of detection, interference, precision of replicates, performance against a specific target nucleic acid sequence, or performance against multiple target nucleic acid sequences in a nucleic acid assay; and (iv) selecting one or more of the modified nucleic acid tag sequences used in step (iii) for use in a nucleic acid assay.

The invention further includes a method as described above, wherein the modification in step g) sub-step (i) comprises systematically deleting nucleotides from the tag sequence.

The invention further includes a method as described above, wherein the performance characteristic(s) is selected from the group consisting of one or more amplification performance characteristic(s); interference with nucleic acids in the nucleic acid assay; interference with one or more oligonucleotides in the nucleic acid assay; interference with one or more target nucleic acids in the nucleic acid assay; interference with one or more amplicons in the nucleic acid assay; assay reproducibility; or quantification.

The invention further includes a method as described above, wherein the performance characteristic is quantification.

The invention further includes a method as described above, wherein the quantification is real-time quantification.

The invention further includes a method as described above, wherein the quantification is end-point quantification.

The invention further includes a method as described above, wherein the performance characteristic is a dynamic range for detecting target nucleic acid; limit of detection; precision of replicates; or reaction kinetics.

The invention further includes a method as described above, wherein the performance characteristics comprise reaction kinetics.

The invention further includes a method as described above, wherein a nucleic acid sequence in the pool is used as a tag in a nucleic acid assay and reduces interference with a nucleic acid in the nucleic acid assay to about 95% or less compared to the amount of interference present in an untagged assay.

The invention further includes a method as described above, wherein a nucleic acid sequence in the pool is used as a tag in an in vitro nucleic acid assay and accelerates reaction kinetics to about 105% or more compared to the reaction kinetics in an untagged assay; slows reaction kinetics to about 95% or less compared to the reaction kinetics in an untagged assay; increases sensitivity for a target nucleic acid so that the amount of target nucleic acid needed to obtain a detectable signal is about 95% or less of the amount of target nucleic acid required in an untagged assay; decreases sensitivity for a target nucleic acid so that the amount of target nucleic acid needed to obtain a detectable signal is about 105% or more of the amount of target nucleic acid required in an untagged assay and/or increases replication precision by about 105% or more compared to an untagged assay.

The invention further includes a method as described above, wherein the nucleic acid assay is an in vitro isothermal amplification assay.

The invention further includes a method as described above, wherein the nucleic acid assay is an in vitro PCR amplification assay.

The invention further includes a method as described above, wherein the tag is part of an amplification oligomer.

The invention further includes a method wherein the tag is a barcode tag sequence for a sequencing reaction.

The invention further includes a method wherein the tag is a barcode tag sequence for a single molecule sequencing reaction.

The invention further includes a method as described above, wherein the tagged assay decreases the performance parameter by from 25% to 94%, from 50% to 94%, or from 75% to 94% compared to the untagged assay, wherein each range is inclusive of all whole and partial numbers therein.

The invention further includes a method as described above, wherein the tagged assay increases the performance parameter by from 105% to 150%, from 105% to 200%, or from 105% to 500% compared to the untagged assay, wherein each range is inclusive of all whole and partial numbers therein.

The invention further includes a method as described above, wherein the tag sequence has a Tm that is less than or equal to 72° C.

The invention further includes a method as described above, wherein the tag sequence has a primer dimer energy formation that is less than or equal to −10.0 kcal/mol; the tag sequence has a hairpin stability energy that is less than or equal to −4 kcal/mol; the 3′ region of the tag sequence is less than 80% complementary to the one or more oligonucleotides in the searched database and/or the nucleic acid assay comprises two or more target nucleic acids.

The invention further includes a method as described above, wherein the database having one or more nucleic acid sequences is a collection of various nucleic acid sequences corresponding to a nucleic acid assay, a public collection of nucleic acid sequences, an aligned collection of nucleic acid sequences, the pool of nucleic acid sequences, or a combination thereof.

The invention further includes a method as described above, wherein the database having one or more nucleic acid sequences is a database containing sequence(s) that are derived from: collections of various nucleic acid sequences corresponding to a nucleic acid assay; a public collection of nucleic acid sequences; a collection of aligned sequences, the pool, or a combination thereof.

The present invention also encompasses a nucleic acid tag sequence obtained by any one of the methods as discussed above.

The present invention also encompasses an amplification oligomer having a nucleic acid sequence that includes a tag sequence obtained by any one of the methods discussed above.

The present invention further encompasses a method for identifying nucleic acid tag sequences for use in an in vitro nucleic acid amplification assay, comprising the steps of: a) generating a pool of nucleic acid sequences, wherein the pool is at least three nucleic acid sequences from Table 1; b) screening the pool of nucleic acid sequences against a database containing one or more nucleic acid sequences to identify percent complementarity between nucleic acid sequences in the pool and nucleic acid sequences in the database; c) screening the pool of nucleic acid sequences to determine a performance characteristic selected from the group consisting of: G-C content, multimer formation, primer-dimer formation, Tm, hairpin stabilization energy, self dimer stabilization energy, internal structure formation, G-quartet formation, hybridization energy, activity in an enzyme reaction, and combinations thereof; d) generating a sub-pool of nucleic acid sequences from the results obtained in step b), step c) or steps b) and c); and e) selecting one or more nucleic acid sequences from the sub-pool for use as tag sequences in a nucleic acid assay.

The invention further includes a method as described above, further comprising: f) synthesizing an amplification oligomer containing a tag sequence selected at step e); and g) performing an in vitro nucleic acid amplification reaction using the amplification oligomer.

The invention further includes a method as described above, wherein the sub-pool at step d) contains nucleic acid sequences with Tm values that are within ±2 degrees C. from a mean Tm of nucleic acids in the sub-pool; wherein the sub-pool at step d) contains nucleic acid sequences with Tm values that are within ±5 degrees C. from a mean Tm of nucleic acids in the sub-pool; nucleic acid sequences with Tm values that are within ±10 degrees C. from a mean Tm of nucleic acids in the sub-pool; nucleic acid sequences with G-C contents that are within ±5% from the mean G-C content of the nucleic acids in the sub-pool; nucleic acid sequences with G-C contents that are within ±10% from the mean G-C content of the nucleic acids in the sub-pool; and/or nucleic acid sequences with G-C contents that are within ±30% from the mean G-C content of the nucleic acids in the sub-pool.

The invention further includes a method as described above, wherein the sub-pool at step d) contains nucleic acid sequences with G-C contents from 30% to 80%, from 40% to 70%, or from 30% to 50%.

The invention further includes a method as described above, wherein the sub-pool at step d) consists of the nucleic acid sequences in Table 2.

The invention further includes a method as described above, wherein the sub-pool at step d) contains nucleic acid sequences with lengths from 5 nucleobases to 100 nucleobases.

The invention further includes a method as described above, wherein the in vitro amplification reaction performed at step g) has reduced interference between nucleic acids in the reaction when performed with the tagged amplification oligomer from step f) compared to when performed using an untagged amplification oligomer; the method has reaction kinetics that are accelerated by about 105% or more when performed with the tagged amplification oligomer from step f) compared to when performed using an untagged amplification oligomer; the method has reaction kinetics that are reduced to about 95% or less when performed with the tagged amplification oligomer from step f) compared to when performed using an untagged amplification oligomer; the method has increased sensitivity when performed with the tagged amplification oligomer from step f) compared to when performed using an untagged amplification oligomer, wherein the in vitro amplification reaction using the tagged amplification oligomer requires an amount of starting material that is about 95% or less than the minimum amount of starting material required in an untagged assay in order to obtain a detectable signal; the method has decreased sensitivity when performed with the tagged amplification oligomer from step f) compared to when performed using an untagged amplification oligomer, wherein the in vitro amplification reaction using the tagged amplification oligomer requires an amount of starting material that is about 105% or more than the amount of starting material required in an untagged assay in order to obtain a detectable signal; and/or the method has a replication precision that is about 105% or better when performed with the tagged amplification oligomer from step f) compared to when performed using an untagged amplification oligomer.

The invention further includes a method as described above, wherein the tagged assay decreases the performance parameter by from 25% to 94%, from 50% to 94%, or from 75% to 94% compared to the untagged assay, wherein each range is inclusive of all whole and partial numbers therein.

The invention further includes a method as described above, wherein the tagged assay increases the performance parameter by from 105% to 150%, from 105% to 200%, or from 105% to 500% compared to the untagged assay, wherein each range is inclusive of all whole and partial numbers therein.

The invention further includes a method as described above, wherein the one or more nucleic acid sequences in a database is a collection of various nucleic acid sequences corresponding to a nucleic acid assay, a public collection of nucleic acid sequences, an aligned collection of nucleic acid sequences, the pool of nucleic acid sequences, or a combination thereof.

The invention further includes a method as described above, wherein the one or more nucleic acid sequences in a database contains sequence(s) that are derived from: collections of various nucleic acid sequences corresponding to a nucleic acid assay; a public collection of nucleic acid sequences; a collection of aligned sequences, the pool, or a combination thereof.

The invention further includes a method as described above, wherein the in vitro amplification assay is an isothermal amplification assay; a multiplex amplification assay or a PCR amplification reaction.

The invention further encompasses a tagged amplification oligomer containing a tag sequence obtained by any one of the methods discussed above.

The invention further encompasses a multiplex in vitro amplification reaction mixture containing a tagged amplification oligomer with a tag sequence obtained by any one of the methods discussed above.

The invention also encompasses a multiplex in vitro amplification reaction mixture containing two tagged amplification oligomers, each with a tag obtained by any one of the methods discussed above.

The invention also encompasses a multiplex in vitro amplification reaction mixture, wherein the two tagged amplification oligomers each have a tag with the same nucleotide sequence.

The invention also encompasses a multiplex in vitro amplification reaction mixture containing three or more tagged amplification oligomers, each with a tag obtained by any one of the methods discussed above.

The invention also encompasses a kit for amplification of a target nucleic acid, wherein the kit contains a tagged amplification oligomer containing a tag sequence obtained by any one of the methods discussed above.

The invention also encompasses a kit for amplification of a target nucleic acid, wherein the kit contains at least two tagged amplification oligomers containing, each containing tag sequences obtained by any one of the methods discussed above.

The invention also encompasses a kit according as discussed above, wherein the tag sequences are the same nucleic acid sequence in each tagged amplification oligomer.

The invention also encompasses a collection of nucleic acid sequences useful as tag sequences for use in a nucleic acid assay, wherein the collection contains at least two of the sequences in Table 1, Table 2, or Table 3.

In another embodiment, the method can be applied to sequences intended to be used in uniplex or multiplex assays.

However, in multiplex assays, interference between the multiple target nucleic acids, from which at least a part of each is intended to be amplified or detected, can cause reduced and inaccurate measurement of the amount of the target nucleic acid in the reaction mixture.

To address this issue, the present disclosure provides a semi-quantitative method that allows for effective discrimination between the levels of interference among multiplex systems with different (or same) tag sequences. The method compares the performance of each of the sequences in a uniplex format, against their performance in a duplex format to arrive at a qualitative determination of the utility of a particular set of sequences together in a particular duplex or multiplex reaction. In a further aspect, the tags are ranked semi-quantitatively in order of their observed interference in the multiplex reaction. This information can be used for the studies which will enable a new user to quickly identify and test tag combinations for a new multiplexed amplification system, and ultimately determine an improved reaction mixture for nucleic acid assays.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A and 1B. Schematic flow charts illustrating a screen for universal Tags. FIG. 1B illustrates that the random oligonucleotide sequences from FIG. 1B are preferably screened in uniplex format and then in duplex format. However, this is non-limiting. The random oligonucleotide sequences can be screened serially in uniplex then duplex formats, or concurrently in uniplex and duplex formats, or in duplex format without a uniplex screen, or in any arrangement of uniplex and/or multiplex formats.

FIG. 2. In silico screen: Illustrative blast filtered screen of two exemplary random oligonucleotide sequences. in this illustration, the blast screen was designed to identify 3′-end dissimilarity, therefore, the sequences selected to advance to the next stage was the sequences that was most dissimilar at its 3′-end to sequences in the blast. The random oligo sequences are represented as the top line in each of the blast alignments.

FIG. 3. In silico screen: this figure illustrates a series of randomly generated oligomer sequences coupled with certain select characteristics. The illustrated characteristics are not limiting. Selections are made based upon the characteristics.

FIGS. 4-10. Each illustrates a uniplex in vitro nucleic acid amplification screen of random oligonucleotide sequences used as tags in a non-T7 amplification oligomer (FIGS. 4-6) or as tags in a T7 amplification oligomer (FIGS. 7-9). The target analyte in each of these assays was PCA3, except FIG. 10 illustrates PCA3 and PSA analytes for comparison. The in vitro nucleic acid assay format used to generate the results shown in FIGS. 4-6 was a RUt TMA nucleic acid assay. The in vitro nucleic acid assay format used to generate the results shown in FIGS. 7-10 was a RUf TMA, which used an amplification oligomer complex in the direct hybridization configuration. In FIG. 7 (continued) bottom panel, a line is drawn from the number 500 to each of the curves representing 500 copies of analyte in the reaction. In FIG. 9 Set T9 panel there the number 10.sup.4 is shown twice, indicating that a couple of the 10.sup.4 reactions were above a set threshold fluorescence amount and a couple 10.sup.4 reactions and all of the 500 copy reactions were under that threshold amount.

FIGS. 11-17. Each figure illustrates a uniplex in vitro nucleic acid amplification screen using random oligonucleotide sequences as tags in non-T7 and T7 amplification oligomers. Both amplification oligomer species (non-T7 and T7) contain a tag sequence. The target analyte was PCA3. The in vitro nucleic acid assays are TMA reaction in the RUt (FIGS. 11-15) or RUf formats (FIGS. 16-17), with RUf format using a direct hybridization amplification oligomer complex (cPRO). In FIG. 14 for the panel showing N28 & T18 tag sequences there are lines drawn from the 10.sup.4 copy number to the tracings representing 10.sup.4 reactions, and from the 500 copy number to the tracings representing 500 copy reactions. IN FIG. 15 for the panel showing the N47 &T9 tag sequences, there is a line drawn from the 500 copy number to the tracings representing the 500 copy reactions.

FIGS. 18-19 show emergence times from a RUf TMA amplification assay using the T21 and U20 tags incorporated into amplification oligomers in a direct-hybridization amplification oligomer complex. The T21 tag and modified versions of the T21 tag were used, wherein the modified tags are shortened by the number of nucleotides in the tag name (e.g. T21-# is shortened by # nucleotide residues). Residues were removed from the 3′end of the tag sequence. The target nucleic acid is PCA3 and is provided in the assay reactions in 10.sup.4 or 10.sup.2 copy number.

FIGS. 20-21 illustrate uniplex RUh TMA assays wherein the Non-T7 amplification oligomers include a tag sequence indicated in each panel. Target nucleic acids are PCA3 (FIG. 20) or PSA (FIG. 21).

FIGS. 22-23 illustrate TMA amplification reactions wherein a reaction is performed in the presence or absence of a potentially interfering nucleic acid containing a tag sequence. In both of FIGS. 22 and 23, the top two panels show amplification of PCA3 or PSA using an amplification oligomer tagged as indicated. No potentially interfering tagged nucleic acid was present. In both of FIGS. 22 and 23, the bottom two panels show a similar amplification as the corresponding top panels, except that amplification was performed in the presence of a potentially interfering tagged nucleic acid. The potentially interfering tagged nucleic acid used in a duplex oligo reaction was the tagged amplification oligomers disclosed in the figure, but not directed to the target (e.g., FIG. 22 bottom left panel=PSA(U20); FIG. 22 bottom right panel=PCA3(N23); FIG. 23 bottom left panel=PSA(U20); FIG. 23 bottom right panel=PCA3(N23)). “NTC” means non-template control and represents a control nucleic acid that is not targeted by uniplex amplification oligomers or the potentially interfering amplification oligomer.

FIG. 24 illustrates in the top two panels a TMA amplification reaction performed in the presence of a potentially interfering tagged nucleic acid (e.g., left top panel is a PCA3 amplification reaction using a U20-tagged non-T7 amplification oligomer in the presence of a U20 tagged non-T7 targeting PSA; and the top right panel is a PSA amplification reaction using a U20-tagged non-T7 amplification oligomer in the presence of a U20 tagged non-T7 targeting PCA3). The bottom two panels illustrate duplex TMA reactions wherein two amplification oligomer sets are provided in each reaction; one directed to PCA3 and one to PSA, and wherein each target analyte is present in the reaction. In these duplex reactions, the amplification oligomer sets each had tagged non-T7 amplification oligomers and each used the U20 tag sequence. The concentration of target was 10.sup.6 PSA and 10.sup.3 PCA3 for the bottom left panel, and 10.sup.3 PSA and 10.sup.6 PCA3 for the bottom right panel.

FIGS. 25-26 illustrate a semi-quantitative analysis for determining interference, which can be caused by any of a number of components in the amplification system. Lower interference values indicate that the tagged nucleic acid used in that system performed better in that system than did other tags. Top panels represent a duplex oligo reaction as described for FIG. 22-23 bottom panels. Bottom panels represent a multiplex amplification reaction as described for FIG. 24 bottom panel. The emergence time for each reaction condition to reach 10,000 fluorescent units is determined, and then an interference value (I-value) is calculated for each as the sum of the difference between the duplex oligo condition and the corresponding multiplex condition. In FIG. 25, the tagged nucleic acids are the non-T7 amplification oligomers and are both U20 tags. In FIG. 26, the tagged nucleic acids are the non-T7 amplification oligomers, with the PCA3 non-T7 being tagged with N54 and PSA being tagged with U20.

FIG. 27 illustrates a number of values obtained for tagged amplification oligomers used in a series of TMA assay as described in FIGS. 22-26.

FIGS. 28 and 30 illustrate the target capture oligomers, blocker oligomers, amplification oligomers and torch detection probes used an a series of triplex amplification reaction containing varied amounts of analytes as indicated in the figures. The top set of oligomer in FIG. 28 targets PCA3, the middle set target PSA and the bottom set target an internal control sequence. In a first set of reactions, the oligomers in FIG. 28 were used against analytes in amounts also as indicated in FIG. 28, and the amplification oligomers all used U20 tag sequences. In a second set of reactions, a set of oligomers substantially identical to those shown in FIG. 28, except that the U20 tag sequence in the non-T7 targeting PCA3, was substituted with an N54 tag sequence. Exemplary results for the full U20 tagged reactions and for the U20/N54 tagged reactions are illustrated in FIGS. 29 and 31.

FIGS. 29 and 31 illustrates reaction curves for amplification reactions using amplification oligomers having one of the tag sequences selected according to the reactions illustrated in FIGS. 28 and 30.

FIG. 32. T2 ERGa: Comparison of standard u20 with N42 Tag (RUh TMA)

FIG. 33. Triplex RUh TMA reaction containing T2 ERGa/PSA/Internal Control, wherein the all non-T7 amplification oligomers contain a N42 Tag.

FIG. 34 illustrates quantitation data from the T2 ERGa/PSA/IC triplex RUh TMA reaction using N42 tagged non-T7 amplification oligomers from FIG. 33. “Cal” means calibrator and “CON” means control sample.

FIG. 35. Triplex RUh TMA reaction containing T2 ERGa/PSA/Internal Control, wherein the non-T7 amplification oligomers contain N42/N6/N42, respectively.

FIG. 36 illustrates quantitation data from the T2 ERGa/PSA/IC triplex RUh TMA reaction using the N42/N6/N42tagged non-T7 amplification oligomers from FIG. 35. “Cal” means calibrator and “CON” means control sample.

FIG. 37. Triplex RUh TMA reaction containing T2 ERGa/PSA/Internal Control, wherein the non-T7 amplification oligomers contain N42/N15/N42, respectively.

FIG. 38. T2 ERGa/PSA/IC (N42/N6/N42) Quantitation

DETAILED DESCRIPTION

In nucleic acid assays which use tags, the selection of the right tag or combination of tags is important. The presently disclosed methods can be applied to various nucleic acid assays, but are particularly referenced in regard to nucleic acid amplification and sequencing. However, such reference is not intended to limit the scope of the application of the disclosed methods and sequences in any way.

Definitions

Unless otherwise described, scientific and technical terms used herein have the same meaning as commonly understood by those skilled in the art of molecular biology based on technical literature, e.g., Dictionary of Microbiology and Molecular Biology, 2nd ed. (Singleton et al., 1994, John Wiley & Sons, New York, N.Y.), or other well known technical publications related to molecular biology. Unless otherwise described, techniques employed or contemplated herein are standard methods well known in the art of molecular biology. To aid in understanding aspects of the disclosed methods and compositions, some terms are described in more detail or illustrated by embodiments described herein.

“Activity in an Enzyme Reaction” is used herein to refer to a number of enzyme driven functions. The term includes binding, extension, cleavage, recombination, repair, and transcription, when these functions are performed by an enzyme.

“Amplification” of a nucleic acid as used herein refers to the process of creating in vitro nucleic acid sequences that are substantially identical or substantially complementary to a complete or portion of a target nucleic acid sequence. The in vitro created nucleic acid sequences are referred to as “amplification product” or “amplicon” and may include one or more tag sequences or the complement of one or more tag sequences. The tag sequences are incorporated into amplification product using tagged amplification oligomers. Alternatively, the tags can be chemically incorporated into a nucleic acid sequence.

“Amplicon” or the term “Amplification Product” as used herein refers to the nucleic acid molecule generated during an amplification procedure that is complementary or homologous to a target sequence contained within a target nucleic acid. These terms can be used to refer to a single strand amplification product, a double strand amplification product or one of the strands of a double strand amplification product. Using an amplification oligomer comprising a target hybridizing sequence and at least one other region that incorporates into the extension product, results in an amplification product comprising the nucleic acid sequence that is homologous and/or complementary to the amplified portion of the target nucleic acid sequence and the incorporated regions of the amplification oligomer. Incorporated regions forming part of an amplification product include tag sequences.

“Amplification Oligomer” or “Amplification Oligonucleotide” as used herein refers to a nucleic acid oligomer that is used for generating complementary strands from a target sequence of a target nucleic acid. The complementary strand can be made by elongation of 3′-end of an amplification oligomer, as is common when using primers, or can serve as a recognition site for an enzyme to initiate complementary strand synthesis, as is common when using promoter sequences. Amplification oligomers include primers, promoter-based amplification oligomers, promoter primers (which allow for elongation for their 3′-ends and transcription from their promoter sequences), and promoter-providers (which are modified to prevent elongation of their 3′-ends but allow for promoter-driven transcription). Amplification oligomers as described herein may further comprise tag sequences.

Amplification oligomers may be directly or indirectly joined one to another to form an “Amplification Oligomer Complex.” Typically joined together are first and second amplification oligomers targeting opposite binding sites of a target sequence. In this configuration, the first amplification oligomer of the complex hybridizes to a binding site on a target sequence, while the second amplification oligomer does not hybridize to the target sequence. The amplification oligomers may be joined one to the other using a connecting compound such as a C9 linker, an oligonucleotide that hybridizes to portions of each amplification oligomer or other such manners. Alternatively, the amplification oligomers may be joined one to the other by hybridizing together complementary portions of the amplification oligomers. Amplification oligomer complexes can comprise any combination of primer or promoter based amplification oligomer. See US Pat. Pub. No. 2008-0305482 A1 and U.S. patent application Ser. No. 12/828,676 disclosing exemplary Amplification Oligomer Complexes.

The term “barcode” is used herein to refer to a tag sequence incorporated into a nucleic acid allowing for identification of some feature of the nucleic acid. A feature of the nucleic acid includes SNPs. For example, the amplicon species in a SNP analysis are substantially identical except for the SNP. Two separate species of primers can be configured to have a 3′ end that is complementary to one SNP sequence or the other, and to have a unique barcode tag sequence to identify to which SNP species the amplicon corresponds. Detection of the SNP species can then be made by identifying the corresponding barcode. A feature of a nucleic acid includes the sample from which the nucleic acid originated. For example, a plurality of samples can be analyzed for the presence of a target nucleic acid. Each sample can be independently barcoded by performing an amplification reaction to integrate a barcode tag into the sample target nucleic acid. The samples are then combined and subjected to subsequent analysis, which can be an additional amplification or a detection reaction. The various combined amplicons in the reaction will be substantially identical except for the barcode tag sequences indicating from which sample the amplicon originated. Other features of a nucleic acid can be identified by a barcode, as is understood by ordinarily skilled artisans.

“Complementarity” is the percentage or amount of a sequence that is complementary to a target sequence or other sequence. The present methods implicate different situations where both high and low levels of complementarity are useful and desirable. “Minimal complementarity” as used in this application refers to the desire to achieve the lowest possible amount of complementarity between a sequence (a tag sequence, for example) and other nucleic acid sequences that may be present in a reaction mixture so as to minimize binding/reaction there between. One example is that a tag sequence selected for use in an amplification reaction is selected to minimize its complementarity to other nucleic acids that may be present in the reaction that are not intended to bind to the tag sequence. In nucleic acid assays wherein a tag sequence is selected so that it is not used to generate amplification products, then it may be sufficient for only the 3′-portion of the tag sequence to possess minimal complementarity to other nucleic acid sequence and still function as desired. This could be particularly true in the case of primers, where the 3′-portion of the sequence is important for enzymatic extension. The 3′-portion of a sequence may refer to the 3′half, the 3′ third or the 3′ quarter of the sequence, or even less as long as the proper function of the tagged oligonucleotide is not prevented or impeded. In nucleic acid assays wherein it is desired that a tag sequence not randomly hybridize to a nucleic acid in the reaction, then the entire tag sequence is screened for complementarity to a nucleic acid in the system.

“Consisting essentially of” is used to mean that additional component(s), composition(s) or method step(s) that do not materially change the basic and novel characteristics of the methods and compositions described herein may be included in those methods and compositions. Such characteristics include the method of identifying and selecting nucleic acid tags for use in an assay wherein the tags are selected to reduce or eliminate undesired nucleic acid interactions within an assay.

“Database of Nucleic Acid Sequences” is used to refer to an in silico collection of nucleic acid sequences. The database of nucleic acid sequences can be the pool or sub-pool of tag sequences, or can be a collection of nucleic acid sequences that may be present in a nucleic acid assay. For example, the various nucleic acids corresponding to an amplification reaction to determine whether a blood sample contains HIV can include the amplification oligomers, the target capture oligomers, probe oligomers, positive control oligomers, HIV nucleic acids and blood cell nucleic acids. A database of nucleic acids, then, can be any one or combination of these nucleic acids in the assay system. The database of nucleic acid sequences can be a public collection of nucleic acid sequences, such as the collection kept by the National Institute of Health (GenBank, National Center for Biotechnology Information, U.S. National Library of Medicine, Maryland, USA).

“Dynamic Range” of an assay generally refers to how much a target concentration (e.g. in a sample) can vary and still be deleted and quantified. The smaller the range, the less robust is the assay, sometimes measured by the cycle number vs. log of the measured signal. These layer the dynamic range, the greater is the ability of the assay to detect samples/targets with high and low copy number in the same run.

“Hybridization Energy” is used to refer to a measurement of free energy released during hybridization of two nucleic acid sequences. Primer dimer hybridization, G-quartet hybridization, primer binding, hairpin structure formation, internal structure formation, and nucleic acid probe binding are all examples of hybridizations. Candidate nucleic acid tag sequences are screened for hybridization to determine the suitability of the tag sequence fro a given application in a nucleic acid assay. In some instances, hybridization energy favoring hybridization is preferred, in other instances; hybridization energy disfavoring hybridization is preferred. For example, in a nucleic acid amplification assay wherein the tag sequences are screened for use as part of an amplification oligomer, then the hybridization energy for useful tag sequences includes those that do not hybridize to nucleic acid sequences in the amplification system.

“Interference” during nucleic acid assays is a common recognized problem and can be seen in various aspects of an assay, including target sequence interference, amplicon interference and primer interference. For example, multiplex PCR assays are known to suffer from primer interference and dimer formation that can cause a reduction in PCR amplification efficiency and multiplexity capacity. Such interference issues can be measured by techniques known in the art.

“Isolated” as used herein means that a target nucleic acid is taken from its natural milieu, but the term does not connote any degree of purification.

“Label” refers to a molecular moiety or compound that can be detected or lead to a detectable response, which may be joined directly or indirectly to a nucleic acid probe. Methods of synthesizing labels, attaching labels to nucleic acids, and detecting labels are well known (e.g., Sambrook et al, Molecular Cloning, A Laboratory Manual, 2nd ed. (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989), Chapt. 10; U.S. Pat. Nos. 5,658,737, 5,656,207, 5,547,842, 5,283,174, and 4,581,333). More than one label, and more than one type of label, may be present on a particular probe, or detection may use a mixture of probes in which each probe is labeled with a compound that produces a detectable signal (e.g., U.S. Pat. Nos. 6,180,340 and 6,350,579).

“Limit of Detection” or “Detection Sensitivity” generally refer to the ability of an assay to detect, measure or otherwise test or react with one particular target or objective as distinctive from others, e.g. in a sample. “Limit of Detection” as used herein is a measure of sensitivity of an assay. The detection limit for an analytical procedure can be defined as “the minimum single result which, with a stated probability, can be distinguished from a suitable blank value” and “the point where, with a stated probability, one can be confident that the signal due to the measurement can be distinguished from the instrumental background signal”. For a specific analytical procedure, the LOD can also be defined as “the lowest amount of an analyte in a sample which can be detected but not necessarily quantified as an exact value.” The LOD is generally expressed as the amount of analyte at which the analytical method detects the presence of the analyte at least 95% of the time. The LOD is often used in terms of the level at which detection starts to become problematic. There are a number of potential reasons for this, inclusive of the presence of noise or an unstable baseline, the contribution of interferences to the signal, the affect of analytical blanks, and losses during the extraction, isolation or cleanup process. One of the most important reasons for defining a LOD is to identify where the method performance becomes insufficient for acceptable detection of the target analyte, in order that subsequent analytical measurements can stay away from this problematic area. The evaluation of the LOD of an assay is thus critical for trace detection methods, especially where the result will be used for regulatory or public health applications.

“Melting Temperature” as used herein refers to the temperature at which half of the DNA strands are in the double helical state and half of the strands are in a random coil state. The desired melting temperature of a candidate tag sequence varies depending upon the intended use of the tag and the assay format it will be used in. (See e.g., Donald Voet and Judith G. Voet, Biochemistry (1990); Owczarzy et al., Predicting sequence-dependent melting stability of short duplex DNA oligomers, Biopolymers (1997) 44 (3):217-239; and Breslauer, et al., Predicting DNA Duplex Stability from the Base Sequence. Proc. Natl. Acad. Sci. USA. (1986) 83 (11): 3746-3750.)

“Nucleic acid” as used herein refers to a polynucleotide compound, which includes oligonucleotides, comprising nucleosides or nucleoside analogs that have nitrogenous heterocyclic bases or base analogs, covalently linked by standard phosphodiester bonds or other linkages. Nucleic acids include RNA, DNA, chimeric DNA-RNA polymers or analogs thereof. In a nucleic acid, the backbone may be made up of a variety of linkages, including one or more of sugar-phosphodiester linkages, peptide-nucleic acid (PNA) linkages (PCT No. WO 95/32305), phosphorothioate linkages, methylphosphonate linkages, or combinations thereof. Sugar moieties in a nucleic acid may be ribose, deoxyribose, or similar compounds with substitutions, e.g., 2′ methoxy and 2′ halide (e.g., 2′-F) substitutions. Nitrogenous bases may be conventional bases (A, G, C, T, U), analogs thereof (e.g., inosine; The Biochemistry of the Nucleic Acids 5-36, Adams et al., ed., 11^(th) ed., 1992), derivatives of purine or pyrimidine bases (e.g., N⁴-methyl deoxygaunosine, deaza- or aza-purines, deaza- or aza-pyrimidines, pyrimidines or purines with altered or replacement substituent groups at any of a variety of chemical positions, e.g., 2-amino-6-methylaminopurine, 0⁶-methylguanine, 4-thio-pyrimidines, 4-amino-pyrimidines, 4-dimethylhydrazine-pyrimidines, and 0⁴-alkyl-pyrimidines, or pyrazolo-compounds, such as unsubstituted or 3-substituted pyrazolo[3,4-d]pyrimidine (e.g. U.S. Pat. Nos. 5,378,825, 6,949,367 and PCT No. WO 93/13121). Nucleic acids may include “abasic” positions in which the backbone does not have a nitrogenous base at one or more locations (U.S. Pat. No. 5,585,481, Arnold et al.), e.g., one or more abasic positions may form a linker region that joins separate oligonucleotide sequences together. A nucleic acid may comprise only conventional sugars, bases, and linkages as found in conventional RNA and DNA, or may include conventional components and substitutions (e.g., conventional bases linked by a 2′ methoxy backbone, or a polymer containing a mixture of conventional bases and one or more analogs). The term includes “locked nucleic acids” (LNA), which contain one or more LNA nucleotide monomers with a bicyclic furanose unit locked in an RNA mimicking sugar conformation, which enhances hybridization affinity for complementary sequences in ssRNA, ssDNA, or dsDNA (Vester et al., 2004, Biochemistry 43(42):13233-41).

“Oligonucleotide” and “Oligomer” are interchangeable terms and used herein refer to nucleic acid polymers generally made of less than 1,000 nucleotides (nt), including those in a size range from about 5-200 nucleotides in length those having lower limit of about 2 to 5 nt and an upper limit of about 500 to 900 nt, or a lower limit of 5 to 15 nt and an upper limit of 50 to 500 nt, or a 10 to 20 nt lower limit and a 25 to 150 nt upper limit. Preferred oligonucleotides are made synthetically by using any well known in vitro chemical or enzymatic method, and may be purified after synthesis by using standard methods, e.g., high-performance liquid chromatography (HPLC).

“Performance Characteristic” means a characteristic of a nucleic acid tag sequence. A performance characteristic of a nucleic acid tag sequence includes a characteristic that is unique to the tag sequence by itself. This includes, but is not limited to, the length of a tag sequence, the G-C content of the tag sequence, the nucleobase composition of the tag sequence, the melt temperature of the tag sequence, etc. A performance characteristic of a nucleic acid tag sequence also includes a characteristic of an oligonucleotide sequence containing that tag. A performance characteristic can be independent of a nucleic acid assay, or a performance characteristic can be determined with regard to a nucleic acid assay. For example, a performance characteristic that is a hybridization event can be determined for a nucleic acid assay, wherein the hybridization is the formation of a primer dimer. Determination of performance characteristics can be in silico or it can be through wet chemistry.

“Pool of Nucleic Acid Sequences” or “Pool of Nucleic Acids” is used to refer to a collection of nucleic acid tag sequences that will be subjected to an analysis to determine at least one performance characteristic. The pool will have at least three nucleic acids. The pool can be an in silico collection of tag sequences, such as a database of nucleic acid tag sequences. The pool can also be a collection of the chemical compounds, such as a freezer box containing a plurality of vials each with a different tag sequence within. The pool can optionally include other information associated with the individual tag sequences, such as a reference name or a property of the chemical compound.

“Precision” in relation to assays, like PCR, generally refers to the variability (i.e. the lack of) among repeated measurements or observations. Precision can be affected by many different factors.

“Precision of Replicates” refers generally to the ability of the assay to produce the same replicates in the same quantities, often statistically measured by means of standard deviations, such as a t test.

“Primer” or “Non-Promoter Primer” is an amplification oligomer that does not comprise a promoter sequence. Thus, primers comprise at least a target hybridizing sequence that is configured to be substantially complementary to part of a target sequence. The target hybridizing sequence need not have 100% complementarity to its intended binding site, but instead may contain 1 or more of a mismatch, insertion, deletion or modification relative to its binding site, so long as the primer's target hybridizing sequence hybridizes to its binding site under stringent conditions. Primers may further comprise tag sequences, capture sequences, self-complementary sequences for forming hairpin loops, and other sequences in addition to the target hybridizing sequences.

“Probe,” “Detection Probe” or “Detection Oligonucleotide” as used herein refers to a nucleic acid oligomer that hybridizes specifically to a target sequence in a nucleic acid, or in an amplified nucleic acid, under conditions that promote hybridization to allow detection of the target sequence or amplified nucleic acid. Detection may either be direct (e.g., a probe hybridized directly to its target sequence) or indirect (e.g., biotin/streptavidin reporter). Probes may be DNA, RNA, analogs thereof or combinations thereof and they may be labeled or unlabeled. A probe may comprise target-specific sequences and other sequences that contribute to the three-dimensional conformation of the probe (e.g., U.S. Pat. Nos. 5,118,801; 5,312,728; 6,849,412; 6,835,542; 6,534,274; and 6,361,945; and US Pub. No. 20060068417).

“Promoter Primer” is an amplification oligomer that is similar to a primer except that the oligomer further comprises a promoter sequence. Promoter primers are capable of 3′ extension by a polymerase and supply a promoter sequence for transcription by a polymerase. Preferred promoter sequences include promoter sequences recognized by RNA Polymerases, such as sp6 promoters, T3 promoters, T7 promoters and others. For example, the T7 promoter sequence is 5′-aatttaatacgactcactatagggaga. Promoter-primers, therefore, comprise a target hybridizing sequence joined at its 5′-end to a promoter sequence. Promoter primers may further comprise tag sequences, capture sequences, self-complementary sequences for forming hairpin loops and other sequences. These additional sequences may be joined to the 5′-end of the promoter sequence, or they may be joined to the 3′-end of the promoter sequence, thereby being flanked on each end by the promoter sequence and the target hybridizing sequence.

“Promoter Provider” is an amplification oligomer that is similar to a promoter primer except that the 3′-end of the oligomer is modified to prevent elongation by a polymerase. Promoter providers supply a promoter sequence for transcription by a polymerase. Promoter providers may further comprise tag sequences, capture sequences, self-complementary sequences for forming hairpin loops and other sequences. These additional sequences may be joined to the 5′-end of the promoter sequence, or they may be joined to the 3′-end of the promoter sequence, thereby being flanked on each end by the promoter sequence and the target hybridizing sequence.

“Quantization” or “Quantification” is used when referring to accuracy in quantification, precision in quantification, and limit of quantification. Quantification can be end point quantification.

“Speed of Reaction” as used herein can encompass a variety of reaction characteristics by a variety of factors, including the reporter dye, nucleotide and primer concentration, enzymatic activity, and the like (see e.g. Lui and Saint, Analytical Biochemistry 302, 52-59 (2002)). Various mathematical models exist that can describe the reaction kinetics. (see e.g. King et. al., Bio Techniques 47:941-949 (November 2009)).

“Region” as used herein refers to a portion of a nucleic acid wherein said portion is smaller than the entire nucleic acid. For example, when the nucleic acid in reference is an oligonucleotide promoter provider, the term “region” may be used refer to the smaller promoter portion of the entire oligonucleotide. Similarly, and as example only, when the nucleic acid is a target nucleic acid, the term “region” may be used to refer to a smaller area of the nucleic acid, such as the target sequence, an oligomer binding sequence within the target sequence, or the like.

“Relative fluorescence unit” (“RFU”) is an arbitrary unit of measurement of fluorescence intensity. RFU varies with the characteristics of the detection means used for the measurement.

“Sequencing” as used herein refers to methods for determining the precise nucleotide sequence of a target nucleic acid. Various sequencing methods are known including chain termination sequencing, dye terminator sequencing, sequencing by ligation, sequencing by synthesis, sequencing by hybridization, circular consensus sequencing, and single molecule sequencing. So-called next generation and third generation sequencing methods are designed to sequence numerous target templates in parallel. Such methods are particularly useful when the target nucleic acid is a heterogeneous mixture of variants, such as is often the case in a sample from a patient infected with a virus, such as HIV or HCV wherein the patient's viral load typically is a mixed population of a majority species and numerous minority species. Amongst the many advantages, sequencing variants in parallel provides a profile of drug resistant mutations in the sample, even drug mutations present in relatively minor proportions within the sample.

Some next generation sequence methods amplify by emulsion PCR. A target nucleic acid immobilized to beads via a capture probe provides a suitable starting material for emulsion PCR. The beads are mixed with PCR reagents and emulsion oil to create individual micro reactors containing single beads (Margulies et al., Nature. 15 Sep. 2005; 437(7057):376-80). The emulsion is then broken and the individual beads with amplified DNA are sequenced. The sequencing can be pyrosequencing performed for example using a Roche 454 GS FLX sequencer (454 Life Sciences, Branford, Conn. 06405). Alternatively, sequencing can be ligation/detection performed for example using an ABI SOLiD Sequencing System (Life Technologies, Carlsbad, Calif. 92008). In another variation, target nucleic acids are eluted from the capture probe and immobilized in different locations on an array (e.g., the HiScanSQ (Illumina, San Diego, Calif. 92121)). The target nucleic acids are amplified by bridge amplification and sequenced by template-directed incorporation of labeled nucleotides in an array format (Illumina). In another approach, target nucleic acids are eluted from the capture probe and single molecules are analyzed by detecting in real-time the incorporation nucleotides by a polymerase. The nucleotides can labeled nucleotides that release a signal when incorporated (e.g., Pacific Biosciences, Eid et al., Sciences 323 pp. 133-138 (2009)) or unlabeled nucleotides, wherein the system measures a chemical change upon incorporation (e.g., Ion Torrent Personal Genome Machine (Life Technologies, Carlsbad, Calif. 92008)).

As a non-limiting example of identifying and selecting tags for use in a sequencing reaction, the following describes identifying and selecting barcode tag sequences for amplification and detection of majority and minority HIV sequences in a single sample. Human Immunodeficiency Virus (HIV) typically exists in a sample as both majority and minority species. Minority species are often undetected in a sample because of their low prevalence (e.g., 0.5% of total HIV population in the sample). Even with assays that are sensitive enough to detect the minority species, the generic nature of many assays hinders resolution of the various species (e.g., a PCR assay may detect but not differentiate between the species). Minority species are often drug resistant species. A failure to detect the minority species is then a failure to identify an important component of the tested sample. Retroviral therapy is then selected and administered without knowledge of the drug resistant species, thereby selecting for a drug resistant HIV population in the patient.

Sequencing assays, including single molecule sequencing assays, are useful analysis techniques for identifying both the majority and the minority species in a sample because the sequence analysis will provide a population of sequence results representing the majority species and a population of sequence results representing the minority species. Often in sequencing assays, though, the minority species are masked by sequencing errors, which are common with these types of assays.

In order to overcome the error rate problem encountered in sequencing assays and accurately identify minority HIV species in a sample, one can design a barcoded amplification oligomer that is configured to amplify the majority species and a separate barcoded amplification oligomer that is configured to amplify the minority species. The majority HIV species barcoded amplification oligomer can include a 3′ nucleic acid residue that is complementary to and hybridizes with the SNP nucleobase that is associated with the majority species. The minority HIV species barcoded amplification oligomer can include a 3′ nucleic acid residue that is complementary to and hybridizes with the SNP nucleobase that is associated with the minority species. The barcode sequence of the majority species amplification oligomer is unique when compared to the barcode sequence of the minority species amplification oligomer. Furthermore, these barcode sequences are selected using a performance characteristic profile that includes providing a unique identifier despite the error rate associated with single molecule sequencing. Typically, but not necessarily or exclusively, this performance characteristic is length and/or nucleobase composition. The length and/or nucleobase composition of the barcodes are preferably configured with the error rate of the sequencing platform in mind, thereby being able to buffer any errors from masking the presence and uniqueness of the barcode sequences. The majority amplification oligomer and the minority amplification oligomer are then used with a common reverse amplification oligomer, and an amplification reaction can be performed. The amplicons generated from the reaction will include one of the two barcode tag sequences, depending on from which HIV species the amplicon was derived. The amplicons can then be sequenced and the data analyzed. Sequencing errors will likely be present and mask the SNP site; however, the unique barcode sequences that were incorporated into the amplicons based upon the HIV species from which they derived will provide an identification of the SNP feature for species. Thus, either or both of the barcode tag sequences and SNP residue are good endpoints for detection of the HIV species in the tested sample.

In the tag identification method for this example, barcode tags would be selected for use in amplification oligomers that are configured to generate an amplification product from one or another SNP corresponding to different species of HIV. A pool of nucleic acid sequences would be generated and that pool screened for two or more performance characteristics useful for the HIV sequencing reaction. Preferably, but not necessarily or exclusively, the performance characteristics include length and/or nucleobase composition. At least one barcode tag sequence would then be selected for each of the amplification oligomers (i.e., the majority species amplification oligomer and the minority species amplification oligomer) and the barcode tagged amplification oligomers would be synthesized. One or more performance characteristics can then be measured for the various combinations of barcode tagged amplification oligomers. Two or more barcode tagged amplification oligomers can be used in a sequencing assay. Sequencing data can then be analyzed and the presence or absence of various HIV species in a sample can be determined by the sequence data including the unique barcode sequences. Further, the relative abundance of one species to another can be determined by the sequence data including using the relative abundance of unique barcode sequences, one to the other.

As a non-limiting example of identifying and selecting tags for use in sequencing assays, the following describes identifying and selecting barcode tag sequences for amplification and detection of nucleic acid sequences from two or more samples to be combined and sequenced in a single sequencing reaction. It is often desirable to determine via a sequencing reaction the presence, absence or composition of a target nucleic acid in a number of samples. In one such example, the presence or absence of a T2:ERG fusion can be determined for two or more different patients in a single sequencing reaction using barcoded amplification oligomers. In such an example, a sample from a first patient can be amplified to incorporate a barcode sequence. Separately, a sample from a second patient can be amplified to incorporate a barcode sequence that is unique from the barcode used in the first patient's sample. Following amplification and preparation of the amplicons for a sequencing reaction, the samples can be combined and analyzed by sequencing. Resultant sequencing data can then be identified via the barcode tag sequences as having come from amplicons of the first sample or of the second sample.

In the tag identification method for this example, barcode tags would be selected for use in amplification oligomers that are configured to generate an amplification product of the same target nucleic acid, but from separate samples, and then combined for analysis using a sequencing assay. A pool of nucleic acid sequences would be generated and that pool screened for two or more performance characteristics useful for the combined sequencing reaction. Preferably, but not necessarily or exclusively, the performance characteristics include length and/or nucleobase composition. At least one barcode tag sequence will be selected for the amplification oligomers used in a sample and at least one barcode tag sequence that is/are unique from those in the first sample will be selected for the amplification oligomers used in the second sample. The barcode tagged amplification oligomers would be synthesized, and one or more performance characteristics can then be measured for the various combinations of barcode tagged amplification oligomers. Two or more barcode tagged amplification oligomers can be used in a sequencing assay. Sequencing data can then be analyzed for the presence, absence or composition of the T2:ERG target nucleic acid sequence from each sample by using the sequence data including the unique barcode sequences.

“Sub-Pool of Nucleic Acid Sequences” is used to refer to a collection of nucleic acid tag sequences that have been subjected to an analysis to determine at least one performance characteristic, and that are selected for incorporation into at least one oligonucleotide sequence for use in a nucleic acid assay. The sub-pool can be an in silico collection of tag sequences, such as a database of nucleic acid sequences. It is not necessary that the sub-pool is a separate database from the pool database, but instead the sub-pool can be a sub-collection within the larger pool database that is somehow differentiated. For example, the sub-pool can be a smaller collection of tag sequences within the pool, wherein the smaller collection of tag sequences share a common melt temp range. The sub-pool can also be a collection of the chemical compounds, such as a freezer box containing a plurality of vials each with a different tag sequence within. Again, it is not necessary that the sub-pool is physically separate from the pool.

“Tag” as used herein is a user-selected nucleic acid sequence that does not hybridize to the target nucleic acid. A tag sequence can be tailored for a specific assay against a specific target sequence or can be designed to apply to a wide variety of assay formats and/or target sequences. One or more tags can be used in an assay. When two or more tags are used in an assay, the tag sequences can be different from one another or two or more tags in the assay can have substantially the same sequences. Tags can serve a number of functions in nucleic acid-related assays, including but not limited to, amplification oligomers, adapter sequences (e.g. SMRTbell), hairpin adapter sequences, sequencing primers, barcodes, detection sequences, displacer sequences, binding site sequences, sequencing primer binding sequence, stem-loop adapters to circularize a double stranded DNA, capture sequences and the like. Tag sequences may be selected to cause minimal undesired interference in the assay system using the disclosed methods. Tags are incorporated into a nucleic acid sequence enzymatically (e.g., using a polymerase to extend a tagged oligomer) or chemically (e.g., using an enzyme free reaction to attach a tag to a nucleic acid). The tags obtained by the presently disclosed methods may be applied for multiple purposes. For instance, in the TMA format, tags can be “joined” to (either directly or indirectly) or incorporated into a primer (or promoter primer, promoter provider, etc.) to form a tagged primer (or promoter primer, promoter provider, etc.). When used in an amplification reaction, these tagged amplification oligomers will introduce the tag sequence into an amplification product. Subsequent rounds of amplification can include amplification oligomers having target hybridizing sequences that hybridize to all or a portion of the incorporated tag. Incorporating tag sequences allows users to control certain aspects of amplification or multiplex amplification reactions (e.g., primer dimer formation, primer efficiency variations, and other aspects). Tagged amplification oligomers and amplification reactions are described more fully in United States Application Publication No. 2008-0305482, the subject matter of which is herein incorporated by reference in its entirety.

“Target Capture Oligomer”, “Capture Oligonucleotide”, or “Capture Probe” refers to a nucleic acid oligomer comprising at least two regions; a target hybridizing region and a support binding region. The target hybridizing region can be configured to specifically hybridize a target sequence of a target nucleic acid, or it can be configured to non-specifically hybridize to numerous nucleic acids in a sample. Specific and non-specific target capture oligomers are described in U.S. Pat. No. 6,116,078 and PCT Publication Number WO 2008/016988. The support binding region is configured to hybridize with a solid support. Preferably, the solid support comprises a complementary binding member that binds with the support binding region of the target capture oligomer. These complementary members can be nucleic acids, proteins or other complementary binding members. For example, the support binding region can be a nucleic acid and the complementary binding member of the solid support is a nucleic acid that is substantially complementary to the support binding region, thereby allowing for hybridization under a set of conditions. An exemplary nucleic acid support binding region is a substantially homopolymeric tail of about 10 to 40 nucleotides (e.g., A₁₀ to A₄₀ or, T₃A₁₄ to T₃A₃₀, or T₀₋₃A₁₄₋₄₀). The complementary member, then, is substantially complementary to all or a portion of the nucleic acid support binding region (e.g., A₀₋₃T₁₄₋₄₀. The complementary member is attached to a solid support, for example, using a covalent linker, ionic interaction, or chelation. Solid supports include nitrocellulose, nylon, glass, polyacrylate, mixed polymers, polystyrene, silane, polypropylene, metal, or other compositions, of which one is magnetically attractable particles.

“Target Nucleic Acid” as used herein is a nucleic acid on which an analytical assay is focused. In a diagnostic assay the target nucleic acid is the nucleic acid that, if present in a sample, indicates the presence of the corresponding nucleic acid of interest or organism. In an antisense RNA assay, the target nucleic acid is the mRNA that is targeted by the antisense oligomer. Target nucleic acids comprise one or more target sequences. Target nucleic acids may be DNA or RNA and may be either single-stranded or double-stranded. The target nucleic acid may include other sequences besides the target sequence. Typical target nucleic acids include virus genomes, bacterial genomes, fungal genomes, plant genomes, animal genomes, rRNA, miRNA, tRNA, or mRNA from viruses, bacteria or eukaryotic cells, mitochondrial DNA, or chromosomal DNA.

“Target Specific Sequence” or “Target Hybridizing Sequence” as used herein refers to an oligonucleotide sequence, which is configured to stably hybridize to a portion of the target sequence. In one embodiment, the target specific sequence is fully complementary with the target sequence, and contains no mismatches. In another embodiment, the target specific sequence is complementary to the target sequence and stably hybridizes to the target sequence under stringent conditions, but contains 1 or 2 or 3 or 4 or 5 mismatches. In one embodiment, the target specific sequence includes at least 10 to as many as 50 nucleotides which are complementary to the target sequence.

“Template Sequence” or “Target Sequence” as used herein is a sequence within a target nucleic acid on which an analytical assay is focused. For example, if the assay is focused on amplification of a target nucleic acid, then the target sequence is a part of the target nucleic acid wherein the amplification oligomers are configured to hybridize and generate an amplicon. Similarly, if the assay is a sequencing assay, then the target sequence is the portion of the target nucleic acid that is sequenced. A target sequence can be the entire target nucleic acid or the target sequence can be a portion of the target nucleic acid. Where the target nucleic acid is originally single-stranded, “target sequence” also refers to the sequence complementary to the target sequence as present in the target nucleic acid. Where the target nucleic acid is originally double-stranded, target sequence refers to both the sense (+) and antisense (−) strands. In choosing a target sequence, the skilled artisan will understand that a sequence should be chosen to distinguish between unrelated or closely related target nucleic acids.

Detection of target nucleic acids may be accomplished by using any known method. For example, amplified nucleic acids may be associated with a surface that results in a detectable physical change, e.g., an electrical change, or can be detected using mass spectrometry. Nucleic acids may be detected in solution phase or by concentrating them in or on a matrix and detecting labels associated with them (e.g., an intercalating agent such as ethidium bromide or cyber green). Nucleic acids may be detected using nucleic acid sequencing techniques. Other detection methods use probes complementary to a sequence in an amplified product and detect the presence of the probe:product complex, or use a complex of probes to amplify the signal detected from amplified products (e.g., U.S. Pat. Nos. 5,424,413 and 5,451,503, Hogan et al., U.S. Pat. No. 5,849,481, Urdea et al.). Other detection methods use a probe in which signal production is linked to the presence of the target sequence. In some instances, the probe is degraded by the amplification enzyme to release the fluorophore from the presence of the quencher (e.g., TaqMan, U.S. Pat. No. 5,210,015). In other instances a change in signal results only when the labeled probe binds to amplified product, such as in a molecular beacon, molecular torch, or hybridization switch probe (e.g., U.S. Pat. Nos. 5,118,801 and 5,312,728, Lizardi et al., U.S. Pat. Nos. 5,925,517 and 6,150,097, Tyagi et al., U.S. Pat. Nos. 6,849,412, 6,835,542, 6,534,274, and 6,361,945, Becker et al., US 2006-0068417 A1, Becker et al., and US 2006-0194240 A1, Arnold et al.). Such probes typically use a label (e.g., fluorophore) attached to one end of the probe and an interacting compound (e.g., quencher) attached to another location of the probe to inhibit signal production from the label when the probe is in one conformation (“closed”) that indicates it is not hybridized to amplified product, but a detectable signal is produced when the probe is hybridized to the amplified product which changes its conformation (to “open”). Detection of a signal from directly or indirectly labeled probes that specifically associate with the amplified product indicates the presence of the target nucleic acid that is amplified.

“Transcription Mediated Amplification” refers to an isothermal amplification method wherein at least a portion of the amplification cycle include making RNA transcripts from a target sequence. depending on whether the primer or the promoter based amplification oligomer is designed to hybridize the initial target nucleic acid in a sample, then the TMA assay is referred to as reverse TMA (RTMA) or forward TMA (TMA). When using tag sequences in one or more of the amplification oligomer species in the reaction, the reactions are referred to herein as follows. (a) “Full,” meaning that the amplification oligomer species used in the reaction include a first tagged-amplification oligomer in a 1:1 ratio with the captured target nucleic acids; a second tagged-amplification oligomer in a 1:1 ratio with the captured target nucleic acid; an excess of a first amplification oligomer that targets the complement of the tag sequence of the first tagged-amplification oligomer; and an excess of a second amplification oligomer that targets the complement of the tag sequence of the second tagged-amplification oligomer. A 1:1 ratio of the tagged-amplification oligomers is typically accomplished by including the first tagged-amplification oligomer and the second tagged amplification oligomer in a target capture reagent as an amplification oligomer complex. Amplification oligomer complexes are described in U.S. Published Application No.: 2011-0003305, and include, but are not limited to, direct-hybridization complexes, covalently linked complexes and others. Briefly, a target capture reaction is performed wherein a target capture oligomer and an amplification oligomer complex are both hybridized to a target nucleic acid. The hybridized nucleic acids are then removed from the remaining components of a sample, typically using a solid support like a magnetic bead, optionally followed by one or more wash steps. These removed components are then added to an amplification reaction mixture that contains reagents for performing the amplification reaction. It is understood that for the three-quarters and the half reactions described below, that the 1:1 ratios are typically achieved by including just the tagged amplification oligomer in the target capture reaction, though amplification oligomer complexes comprising a tagged amplification oligomer member and a non-tagged amplification oligomer member can be used. (b) “Three-quarters,” meaning that the amplification oligomer species used in the reaction include a first tagged-amplification oligomer in a 1:1 ratio with the captured target nucleic acids; an excess of second tagged-amplification oligomer; an excess of a first amplification oligomer that targets the complement of the tag sequence of the first tagged-amplification oligomer; and an excess of a second amplification oligomer that targets the complement of the tag sequence of the second tagged-amplification oligomer.(c) “Half” meaning that the amplification oligomer species used in the reaction include a first tagged-amplification oligomer in a 1:1 ratio with the captured target nucleic acids; an excess of a second amplification oligomer; and an excess of an amplification oligomer that targets the complement of the tag sequence of the first tagged-amplification oligomer. As is used herein, RUf means Reverse TMA with the full tagged amplification oligomers format. RUh means Reverse TMA with the half tagged amplification oligomers format. RUt means Reverse TMA with the three-quarters tagged amplification oligomers format. (See e.g., WO 2011/003020, throughout and particularly in the Examples discussing half, full, three-quarter and other tag arrangements in TMA reactions; and see WO 2009/140374, throughout and particularly in the figures wherein the flow of a TMA reaction is illustrated. Both of these documents are incorporated herein by reference.)

A. Description of the Method for Identifying and Selecting Tags for Use in In Vitro Nucleic Acid Assays.

The overall method can be schematically represented as is generally shown in FIG. 1A. The Figures illustrate the general tag identification method by reciting an amplification assay as the in vitro assay for testing tagged nucleic acids. As is made clear in this disclosure, though, the tag identification methods are applicable to a variety of in vitro assays, not just amplification assays. Tags are identified as being useful in an in vitro assay that contains additional nucleic acid sequences. Identification is based upon a number of factors, as described herein. Once identified, the tags can be incorporated into one or more nucleic acids used in the in vitro assay. The steps for tag identification are as follows:

1. Random Oligo Generation:

To begin, a pool of oligonucleotide sequences is obtained or generated (as necessary), typically as a random pool of sequences. This can be performed in a variety of ways, including with the aid of a computer program. If desired, the user can set boundaries at this stage for desired characteristics or properties, such as sequence length and GC content. When a computer is used, this phase of the method can be described as an in silico screen. Sequence length as a parameter or boundary will be set depending upon the desired use of the tag. Lengths can be set, for example, to 25-30 nucleotides. G/C content is the amount of G residues and/or C residues in an oligonucleotide or polynucleotide sequence. G/C content in the Tag sequences is reflected as a percentage of the number of G and/or C residues as compared to the total number of residues in the oligonucleotide. Among other things, the G/C content alters the melting temperature of the sequence. Acceptable G/C content depends on the target nucleic acid under consideration. Typical G/C content can be in the range of 30 to 80%, 40 to 70%, or 30 to 50%.

2. Blast:

The pool is subjected to a search to identify sequences that have minimal sequence complementarity to all known sequences in one or more selected databases to obtain a sub-pool of sequences. An example of a useful tool for this purpose is the BLAST (Basic Local Alignment Search Tool) algorithm available from NCBI (National Center for Biotechnology Information). Sequences selected for the sub-pool are those having complementarity of less than 95%, less than 80%, less than 70% or less than 50% to the nucleic acid sequence in the database.

3. Screen for Oligonucleotide Parameters:

The sub-pool of sequences prepared from the step 2 Blast is then subjected to further screening, which may be in silico screening, for features that could negatively affect their performance in regards to the desired functionality, including melting temperatures, activity in an enzyme reaction, G-C content, hybridization energy, multimer formation, internal structure format, G-quartet formation and hairpin stability. A variety of features can be used for the in silico screening, depending upon the desired use of the tags. For example, if the intended use of candidate sequences is as primers/providers in an amplification reaction, the candidate sequences can be screened for one or more of primer/dimer formation, internal secondary structure, inappropriate melting temperature (Tm) values, cross-reaction or other unwanted interactions with other nucleic acid sequences to be used in an assay (including other tag sequences), and the like. Sequences identified to possess an undue amount of negative attributes are removed from the sub-pool. The following are exemplary parameters that can be screened for at this stage. Primer/dimer formation is a characteristic to be avoided in identifying useful tags for an in vitro amplification assay. Primer-dimer formation capability can be determined using software routinely available to one of ordinary skill in the art, e.g. Oligo Primer Analysis Software from Molecular Biology Insight, and references therein. Some internal structures are also understood to typically be undesirable in a nucleic acid assay system, such as the presence of hairpin loops, G-quartets and other unwanted structures. Such structures can be easily identified by methods known to those skilled in the art. Inappropriate melting temperature (Tm) value is another characteristic to be avoided in useful tags. Cross-reaction or other unwanted interactions with other nucleic acid sequences to be used in a reaction mixture (including other tag sequences) are also to be avoided for useful tags. Cross-reactions to be avoided can be from target nucleic acids or other nucleic acids suspected of being present in a sample and/or assay reaction, such as pathogenic or non-pathogenic organisms, mammalian nucleic acids, contaminating nucleic acids from enzymes, side-products of nucleic acid amplification reactions, etc. Cross-reactions can also occur with sequences intentionally included in the assay mixture. For instance cross reactions can occur between a tag sequence and the target sequence, the tag sequence and the target specific sequence of an oligomer, and the tag sequence and other templates, targets, primers, probes, detection sequences, displacer sequences, binding site sequences, capture sequences etc.

4. Synthesis and In Vitro Assays:

Based on the results from the screening steps described above, performance of candidate sequences can be optimized (if desired) by a cycle of systematic sequence design changes followed by a repeat of one or more of the in silico screen described above for the sub-pool and the in vitro screen discussed above.

The sequences that successfully pass the above two screens are then selected for use in the intended nucleic acid assay, or as a component in a reaction mixture, and subjected to rigorous experimentation to benchmark their activity against the desired performance characteristics. This additional experimentation is generally conducted in vitro, that is, the sequences are synthesized and run in an in vitro assay. Cross reactivity in a system inhibits or considerably degrades the amplification performance. The practitioner can then systematically eliminate tags from the candidate tag pool which cross-react with other tags, amplification oligomers, or particular templates, to obtain a non-biased assay.

In typical multiplex amplification reactions, a variety of undesired “side reactions” could occur that ultimately degrade assay performance. For example, various amplification oligomers can interact with one another or with other sequences within the assay. Commonly, primers and/or providers directed towards one target nucleic acid (or group of target nucleic acids) interact with primers and/or providers directed towards another target nucleic acid (or group of target nucleic acids), causing degraded performance of one, or the other or both amplification systems. This problem typically gets worse as the number of target sequences present in a multiplex reaction increases. This problem can be reduced or solved by incorporating a tag sequence into one or more amplification oligomers in the system. The tagged amplification oligomer(s) will then incorporate their tag sequences into the initial amplification product. Subsequent amplification can take place using amplification oligomer comprising sequences that are configured to hybridize to the incorporated tag regions

However, even if specific primer and/or provider interactions are reduced or eliminated, other interactions that degrade assay performance can still be present. For example, the two tagged primers (or primer and provider) can interact with one another. In the half tagged amplification mode described above, the one tagged primer (for example) can interact with other remaining specific primers and/or providers. Furthermore, tagged primer(s) can interact with other oligos in the amplification reaction, such as probes, blockers and target capture oligomers (TCOs), and/or with target nucleic acid related sequences (such as amplicons). Additionally, in an amplification system using two or more tagged amplification oligomers, subsequent amplification reactions are driven by the same amplification oligomer(s) complementary to the tag sequence. Competition between the various amplification reactions for this limiting resource can be a problem. For example, amplification of a target sequence present in a multiplex reaction at high levels will consume the primers complementary to the tag sequence before amplification of a target sequence present in a multiplex reaction at low levels can “complete” its normal reaction.

One solution to this problem is to create unique tag sequences for some or all of the target sequences in a multiplex reaction, and to design them such that they do not interact with each other, or with any other tag set from different target sequences in the reaction, or with any other sequences in the reaction mixture. In this way, each amplification reaction can proceed independently and degradation of multiplex amplification performance is reduced or eliminated. The identification and selection method of this disclosure can be used to generate such unique tag sets.

B. Description of the Method for Identifying and Selecting Tags Having Minimized Interference in a Multiplex Assay.

The present invention further encompasses a method that draws on the power of combinatorial screen and selection to identify improved tag sequences that reduce or eliminate the problem of interference, which is generally observed in multiplexed amplification reactions. Another advantage of the method is that the criteria set for the screen can be varied to match a desired property that the user wants to screen for in a particular assay system. These criteria can be speed of an amplification reaction of a first target nucleic acid relative to the amplification speed on other target nucleic acids, performance of a tag sequence or tagged oligomer within a give system, lack of interference with or by other nucleic acids in the assay system, etc. The final solution in terms of tag sequence(s), number of tags to use in a system, which nucleic acids comprise tags, arrangement of tags and etc. is derived from a large repertoire of user-defined oligonucleotide tag sequences (as opposed to target-specific sequences). Thus, the method transcends the inherent limitation imposed by amplification systems that use target specific primers for amplification. Moreover, the tags are incorporated into the amplicon and can be utilized to enable downstream applications such as sequencing, signal amplification, microarray analysis, etc. One can envision the selected tags to be modified as signaling probes, adapters for ligation-based assays, as tags in sequencing applications and the like.

The overall method can be schematically represented as shown illustrated in FIG. 1B for using tag sequences in a given amplification format. In a multiplex amplification assay, one or more tags are selected by the following steps: 1-4. Preparation of Library of Tag Sequences: A library of tag sequences is prepared using the method described above for identifying useful tags, namely: 1. Random oligo generation; 2. Blast; 3. Screen for Oligonucleotide Parameters; 4. Synthesis and in vitro Assays After the completion of these steps, further identification of useful tags can be determined using the following steps:

5. Screening in Uniplex:

A library of tag sequences is screened for each target sequence individually in assays, for example in a uniplex nucleic acid amplification format, such as TMA. Each tag sequence is thus evaluated qualitatively or quantatively, based on, for example, the precision between replicates limit of detection, sensitivity, kinetics of reaction and emergence time relative to a standard reference tag, if available, or other appropriate parameters as desired.

6. Screening Using Duplex Oligos:

The tag sequences demonstrating optimal performance from the step 5 uniplex screen are subsequently screened in the presence of oligos of the competing target sequence (duplex oligo screen). This screen is done first without using a tag for the second target sequence, and then including the tag chosen from Step 5 for the second target sequence (this could be the same tag or another unique tag) in order to assess the level of oligo interference existing in a given multiplex amplification. The level of oligo interference for each target sequence is again determined qualitatively or quantitatively relative to their performance in uniplex assays.

Methods for such quantitative or qualitative determination are known to those skilled in the art and include, for example, by comparing Ct values between the uniplex and multiplex reactions, by determining the limit of detection of a particular target nucleic acid in a uniplex reaction and in a multiplex reaction, etc.

7. Screening Using Duplex Oligos and Target Nucleic Acids:

Each tag sequence pair showing minimal oligo interference and sufficient performance in the presence of duplex oligos from step 6 are then screened in the presence of duplex oligos and target sequences to ascertain the level of total interference. More specifically, relatively low copy levels of each target sequence are evaluated in the presence of high copies of the competing target sequence (in two separate conditions) with the tag of each target sequence chosen from the duplex oligos screens. The relative amounts of each target sequence to be used for these interference screens, where the target sequence interference can be observed from the amplification curves might be different for each system and therefore should be chosen on a case-by-case basis. The results of the reactions in steps 6 and 7 are then qualitatively and quantitatively evaluated to determine preferred combinations of tags to be used in an assay given the nucleic acids present in the multiplex reaction mixture.

Such qualitative evaluation methods are known to those skilled in the art and include determining whether or not one target nucleic acid can be detected (at a certain input copy level) in the presence of the other target nucleic acid(s).

8. Interference Analysis:

A novel method to quantitate the interference observed for each combination of tags screened as described above has been developed. With this method, an “interference value”, or “I-value”, is determined for each target sequence in a multiplex reaction, and these I-values are added together to yield the total I-value. For example, in a duplex system, [I-value (total)=I-value (target sequence1)+I-value (target sequence2)].

When using Real-Time TMA I-values can be calculated from emergence times. In a duplex system, for example, the I-value of target nucleic acid 1 is calculated by subtracting the emergence time of a relatively low copy level of target nucleic acid 1 in the absence of target nucleic acid 2 from the emergence time of the same (low) copy level of target nucleic acid 1 in the presence of a relatively high copy level of target nucleic acid 2. In an equivalent manner, the I-value of target nucleic acid 2 is calculated using a relatively low copy level of target nucleic acid 2 in the absence or presence of a relatively high copy level of target nucleic acid 1. The sum of these two I-values yields the total I-value for the given set of tags used. The lower the total I-value, the less interference there is between the tags. FIGS. 25 and 26 illustrate the calculation of I values for amplification assays using different tag in the primers. A wide variety of tags and combinations thereof can be screened and the relative interference levels can be rapidly quantitated using this method.

EXAMPLES

The following examples demonstrate the use of this method to select the best tag sequences for use in a TMA assay format. These examples are intended only to demonstrate the use of the selection method and are not intended to limit the scope of application to only TMA or to only amplification assays.

Example 1: Screen for NT7 Tag Sequences

Step 1—Random Oligo Generation:

A large pool of oligonucleotides 25-30 nucleotides in length are randomly generated. Random oligonucleotide sequences can be determined in a variety of manual or automated methods. One manual method for determining random oligonucleotide sequences of a desired length includes a blinded selection of a series of A, C, T, G and/or U residues. One automated method for determining random oligonucleotide sequences of a desired length includes using an algorithm for randomly selecting a series of A, C, T, G and/or U residues. Many algorithms are freely and commercially available to generate a random pool of nucleotide sequences. Suitable algorithms include those found at http://molbiol.ru/eng/scripts/01_16. html; http://www.faculty.ucr.edu/˜mmaduro/random.htm; http://tandem.bu.edu/rsg.html; http://www2.uni-jena.de/biologie/mikrobio/tipps/rapd.html; and, described in Piva and Principato, In Silico Biology, 6, 0024 (2006). Many other algorithms can also be used. For this example, an initial population of −1000 sequences was generated for screening (see step 2 below). These sequences were selected to vary in their GC content, length and Tm.

Step 2—Blast:

The oligonucleotides generated in step 1 above were then subjected to an in silico screen using the BLAST (Basic Local Alignment Search Tool) algorithm available from NCBI (National Center for Biotechnology Information) website. The basic workflow for each oligonucleotide was as follows:

Using the “BLAST Program Selection Guide” available on the website, “nucleotide blast” (blastn) was chosen to search the nucleotide databases using a nucleotide query. The program “blastn” is specifically designed to efficiently find short alignments between very similar sequences and thus is the best tool to use to find the identical match to a query sequence. In this example, the query sequence was one of the tag sequences identified in step 1. Several different databases are available for search using BLAST, and the “nr” nucleotide database was used in this instance.

The goal of this screen was to identify sequences with minimal complementarity to sequences in the data base other than the desired target nucleic acid, thus minimizing the potential of unwanted cross reactivity with non-target nucleic acids that may be present in the assay reaction mixture. Particular care was taken to screen sequences that may be problematic in a given assay. For example, if a viral assay is being developed, the sequences corresponding to other non-target viruses as well as the non-target regions of the targeted virus were carefully examined.

If a candidate tag sequence yielded greater than about 80% overall complementarity to any sequence in the data base, it was rejected. The value for percentage complementarity used as a cut-off will vary depending on the assay under developed as well as the specific requirements of that assay. The 80% value cited here is only an example of a cut-off. Additionally, candidate tag sequences that yielded greater than 6 to 8 contiguous bases of exact complementarity were also rejected in general. However, as discussed above, exact complementarity in the 3′ portion of the molecule is undesirable if the tag is to function as a primer, for example. Therefore, more exact complementarity than listed above could be tolerated in the 5′ portion of a tag primer candidate in this case, as long as 3′ complementarity was low. FIG. 2 shows an example of such an analysis.

Other rejection criteria can also be set, depending on the particular specifications of the assay under development. Based on the chosen criteria, a fraction of the tag sequence candidates are rejected and the remainder taken on to the next screening step.

Step 3—Screen for Primer Parameters:

A pool of about 100 sequences (Table 1) was identified in steps 1 and 2 above out of an initial population of 1000 randomly generated sequences. These 100 sequences were then subjected to another screen wherein properties that could decrease a tag candidate's effectiveness in the desired application, such are hairpin or primer-dimer formation, were identified and those sequences were removed from the pool. For this second screen, the oligo analyzer software available from Integrated DNA technologies (www.idtdna.com/analyzer/Applications/OligoAnalyzer) was used.

Representative data from this second screen are shown in FIG. 3. Candidate tag sequences that were predicted to form hairpin structures with a stability of −4 kcal/mole or greater (i.e., a more negative value) were discarded. Candidate tag sequences that were predicted to form primer-dimer structures with a stability of −10 kcal/mole or greater (i.e., a more negative value) also were discarded. Candidate tag sequences with Tm values >72° C. were also discarded in this example.

Table 1 presents the results of the screens, wherein the asterisk symbol “*” following a value indicates that the corresponding sequence is discarded as a candidate tag sequence based on that result. Sequences which lack any asterisk are selected as tags.

TABLE 1 Results of Tag Parameter Screens self Hairpin dimer stabili- stabili- zation zation energy energy SEQ maximum maximum Seq ID GC Tm (dG) in (dG) in # NO: Sequence (5′-3′) Length content (° C.) KCal/mol) KCal/mol 1 2 CCCCGTCAAACAAAAACGG 30 56.7 65.9 -4.81 -11.00* GAGCGTGTACC 4 3 CCATAGGCCTTCTGCACTG 30 53.3 63.2 -1.18 -12.47* CTCCATATACC 6 4 GTCCCCATCGGAGGGCATC 30 60 67.5 -3.36 -8.16 TTATCGTGCCT 8 5 CCGCCCTCCTTCGCCCCCC 30 66.7 70.1 -1.67 -9.75 GGTGAAATAAC 12 6 AATGCTCACCTCTATTCGG 30 46.7 60.9 -0.71 -5.13 GACTTGAGTAC 21 7 CCCGCGCACCACCTCCATC 30 66.7 70 -2.19 -10.36* ACGCAGAAGAG 25 8 GTCGGAACGCCAGGTACAG 30 60 66.7 -2.08 -9.89 TTAGCGCATCC 26 9 AAGTCACTGGCCAGCATAA 30 53.3 65.4 -0.76 -16.38* TGCGTGAAGGG 27 10 GTGATGCTTTATGAGATTC 30 50 61.7 -2.15 -9.75 CGGTCTCCGAC 28 11 GACGGTGCATCACCCGCAT 30 60 67.6 -2.79 -7.05 TTGCTGTAGCG 34 12 AGAATTCTTGCAGGTAGAG 30 46.7 62.2 -2.22 -11.71* GTCCCCTCATT 35 13 AAGCCAAAATTACAATCGA 30 40 59.1 1.41 -9.71 TCCCTACCAAC 37 14 ATCTTGCACCTTCCCAGAT 30 50 64.3 0.4 -7.05 GTAAACCCCCT 42 15 GAAGCGGCAGCTCAGCCGG 30 66.7 69.6 -6.97* -9.82 TTCTCGGAGAG 43 16 GCACGCGGGCTCCTTGGGA 30 60 67.1 -0.1 -10.36* CACTATGATTG 61 17 CCCATCAGGACAGTCAGCT 30 56.7 66.5 -1.35 -10.24* GCCCACGAATT 78 18 CTTTAGTGCGGTAGGACCG 30 56.7 64 -5.07 -10.58* AGACTACCGTG 79 19 TTATGTGCCAGCTGGGCCT 30 63.3 69.6 -2.6 -16.38* AAGGCTCCGGG 80 20 GACTCTCCTAGGGCGTTCG 30 63.3 67.3 -0.43 -10.30* TCTGGGACTGC 82 21 CGGAGAATACCCTCGACTG 30 46.7 60.1 -0.83 -6.76 TATCATATCGT 84 22 TTCATCGAGGTACATTGGT 30 40 59.6 -0.18 -6.76 GCTATTCCATT 86 23 TACCACCTGGTTCAAGGTG 30 60 67.9 -3.73 -10.87* TGCCGTACGCG 87 24 AGGAGAACCAGCCTGGAGC 30 53.3 64.8 -1.93 -6.62 GTTTAAGCATC 88 25 GATGTCCTAAAATGAGGCG 30 46.7 60.6 -0.28 -4.67 TGGCAATAGAG 89 26 CAGAGTCATGTATACCCAC 30 50 62.1 -0.19 -6.76 TGTCGGTCGAA 104 27 GTCAGGCTAGGGGGTTATC 30 63.3 68.2 -2.62 -6.14 CCAGCAACGGC 106 28 TGGGTTCTGCTAACCGGTG 30 53.3 65 -2.02 -12.43* CCGTTCTTAAC 116 29 TTTTTGACAGTGATGAAGA 30 43.3 60.7 -1.61 -3.65 GGGAGGTACGA 133 30 GAGAACTCGCGCTCCCTCA 30 56.7 65.4 0.21 -10.36* CTCCGTTTAGA 136 31 CTATGGTTCGTTACTGAAT 30 46.7 61 -1.19 -7.13 CGAAAAGCCGC 138 32 TAGCTATCAAAACAGGCGT 30 43.3 60 -1.05 -8.26 CATCGGTTAAG 145 33 AGGACGCTGACACCGTTGG 30 60 68.1 -4.77* -9.69 GGTAAAGCGTG 152 34 CCTGCTTAGGGTCACTTAA 30 53.3 63.5 -0.37 -9.89 ACTACTGGCGC 155 35 GGTGATGGCCCATACCGAT 30 66.7 70.1 -1.55 -9.28 CACGCCCGCAG 156 36 CGGCAGGAGGGACTGCGAT 30 60 66.5 -2.59 -6.69 TTCCATAGAGC 159 37 TGGCCGGAGAGAGGATAGG 30 60 67.5 -1.62 -9.75 AAGCGGGACTA 161 38 TAGCAGGTGTCTCGGTCCT 30 53.3 64.6 -4.25* -7.05 CAACTGCAAAC 163 39 ACACATCCCAGGACTGCCG 30 60 68.5 -1.8 -9.28 TGGCCTACGTA 171 40 GTGCTAGCCCGGGCCCTTC 30 63.3 69 -3.7 -22.17* TTAACTCGGGA 172 41 CGGAATCTGAACATCTATC 30 53.3 64.5 -3.69 -10.36* AGAGCCGCGCT 174 42 GACGAGCTTGTTCCAATTC 30 56.7 65 -2.66 -9.96 CTCGAGCCGAG 179 43 GTTGGGGAGGGGCACTACG 30 60 67 -1.96 -3.61 ACTTAGGGCTA 182 44 AATGTGGACGGCCGCTCCG 30 56.7 67.3 -3.59 -16.50* TACTTCTGACA 183 45 AGGGCCAGCAGCTGGTTCC 30 60 69.3 -2.32 -10.24* TTCGCCAGTTA 185 46 GGCCGTCAATGTGTTTTGC 30 56.7 67.7 -1.48 -9.75 ACCCAACCGGA 188 47 CAGTGACTGGGCTAGTGAA 30 53.3 62.9 -4.43* -7.81 GTGAGTCACAG 193 48 TCCCACGTCCTTCGACGCA 30 53.3 65.6 -3.02 -6.76 CACTGTAACTT >301 49 TCATGTATCGCCCGTGGGT 25 56 62.3 -0.69 -6.34 AAGCTC >305 50 ATGTTATGGAGAGTGGGTT 25 44 57.9 0.75 -3.14 AGGCAA >307 51 ATGAGGGAGTAAGGAGATT 25 44 55.4 0.57 -2.91 AGGTTC >309 52 CATGCTGCCCGCATACACT 25 64 66.5 -6.62* -14.84* TGCGGG >313 53 GCCCAGCAGTTATACAATT 25 52 60.3 -0.2 -6.21 CGTGGC >314 54 TTGGGCTCTCCAGTAGCCG 25 52 62.2 -2.14 -7.81 AACAAA >316 55 TGACGTTAAACGCAATCCG 25 44 59.4 -3.94 -10.36* CGTAAA >325 56 GTCGCCATTCAGGACACGC 25 56 63.4 -1.92 -10.36* GAAACT >327 57 GTGGTTGCTACAGCCTAGC 25 52 60 -1.79 -5.7 CTAGAT >336 58 CCACTTTTCATTCCGAGTC 25 56 62 -0.08 -10.36* CACGCG >339 59 AGGAGGAACCGGAAGATCT 25 48 57.6 -1.09 -9.75 AATCTG >342 60 CCAATGCTTTCAAATAACC 25 40 56.2 0.57 -3.89 CGTTCT >343 61 GCGACTGTGGCAACCCCAT 25 60 66 -3.65 -8.33 TTCGCA >346 62 AAAAAACGGAGGAGTCGAA 25 48 59.4 -0.75 -6.76 CCTTGG >350 63 AGTTGGATGGATATCTCGC 25 48 59.4 -0.32 -7.06 TCGTGA >356 64 CGCTGTCCTCTCTGACACT 25 52 60 -1 -4.87 AAAGGT >357 65 ATTTCAATAGTCAACCCGG 25 40 56.1 0.44 -9.75 TATCCA >505 66 TTCGCGCCAGCGACCCCAC 25 60 66.3 -3.52 -10.36* TTATGA >510 67 GGTTGGGGGGCTCGGCTCA 25 64 65.2 0.09 -5.38 TGTATC >516 68 ATGATGCTGAATCGCGATG 25 60 65.1 -0.13 -16.46* GGGGGG >517 69 TAAGGAGACTAGGTTCCAA 25 44 55.5 -0.84 -6.34 TAGCTG >523 70 TTACACAAATCGTGGGTTG 25 48 60.6 -1.86 -9.28 GCCTCT >534 71 CGAAAGCGTTCCGCAGGAC 25 64 67.1 -2.4 -6.75 CCCCTT >538 72 CACCCTTGGACACGTGGAA 25 64 65.7 -1.49 -10.20* GTGGGC >541 73 AAAGTCTGAGAATGAGTGA 25 36 53.8 0.92 -3.43 TACCAT >544 74 ATATTGGTAGTTTTGTCCG 25 40 54.9 0.67 -3.91 CTGTAG >549 75 CGGAAGATCTAATCTGCAC 25 44 57.5 -0.78 -7.82 GCAATT >608 76 GCGCCTCGTTGGGCAGAAG 30 53.3 66 -2.36 -9.89 TTTGTGGAAAT >610 77 ATCTTCACCTACCGAGTTC 30 53.3 63.4 -2.39 -9.28 TACGGGCCTAC >613 78 CCCACAACTTGCACCCGCT 30 63.3 68.9 -2.32 -7.05 ATGCGACCCTG >618 79 GCCCAGGAGCTCTCCTGGG 30 63.3 67.5 -7.98* -15.93 TAACAGTAGCG >620 80 CACGGCCCCCAGGCGGCGT 30 70 72.5* -3.3 -9.28 ATCAGGGATGA >623 81 TCCCCGGCACGGACCGCAA 30 70 73.2* -5.63* -9.75 GGGACCAAAGC >629 82 GATTAGTGGCCCAACGGGA 30 50 63.6 -2.04 -9.28 ACAAACTTCCT >701 83 CGCCCGTCCCAGACCCTTA 30 60 66.8 -0.61 -5.02 CTCACTATGGA >703 84 GCTACACGCCAGAGGCGCC 30 66.7 71 -7.27* -16.03* GCTACAGCGAT >706 85 GAGATTGTACCCTACAGTC 30 46.7 60.4 -1.58 -4.26 CGATTACCGAT >715 86 CGCAGTAAAAGGGCACAGG 30 43.3 60.1 -2.05 -19.3 TAATTACCTTA >718 87 AGGGTGTCTTGAACTACTG 30 56.7 67.6 -3.43 -9.89 GCGCAGCCCAT >721 88 CCGCAATCCGGTGACGGCC 30 76.7 74.8* -5.90* -16.50* GGACCGGCAGG >723 89 TCGGCGGCGGGTAGTCAGT 30 66.7 70.3 -3.26 -6.97 TCGCTACCTGG >729 90 CCAGGACTGCCGTGGCCCA 30 70 72.7* -2.93 -9.98 CGCACTCACGA >740 91 TTGACGCAGGCCCCCGGGG 30 66.7 71.2 -2.92 -22.03* CGACTTCATAC >743 92 CGAAAGGAGTTCGAGTGTA 30 56.7 64.7 -3.81 -12.90* TCCGGAAGGCG >745 93 AGGCGCACTGCGACTTAGG 30 70 72.5 -4.68* -22.71* GCTAGCCCCCC >751 94 GATGTGATCTGGACCCTAC 30 60 66.7 -3.51 -7.74 GGGAGGGGACA >754 95 TGGGCTGGGGGAGTGAGTC 30 73.3 74.1* -10.51* -9.31 GCTCCCGCAGC >757 96 AGTCCCAGATATGAGAGAA 30 43.3 60.4 -3.75 -4.39 GCGAAGCATAA >805 97 CGTTTCAGCATCGATGTCC 25 40 55.7 -0.04 -13.62* TAAAAT >815 98 ACTATTACACCACGTACCG 25 48 57.5 -2.43 -6.3 TAGGTC >816 99 GGGCAACACCGCGAGCTAA 25 56 61.9 -0.21 -10.60* TTATCC >818 100 GCGCGCGGCCGAGAATCGT 25 72 69.7 -1.77 -17.11* TGGAGG >826 101 CGCGTCGGGCTTTCGTCTA 25 68 67 -1.3 -10.60* CCCTGG >829 102 GGGCGGCCACCGGGGGACC 25 88 77.7 -3.41 -9.75 CTGCCC U20 tag 1 GTCATATGCGACGATCTCA 20 50 52.8 0.38 -7.82 (Std) G

Step 4—Synthesis and In Vitro TMA Assay:

Based on the screen for primer parameters, about 55-60 candidate sequences were identified as good candidate tag sequences (Table 2).

TABLE 2 Synthesized Universal non-T7 primer tags for in vitro experimentation Se- SEQ Tag quence ID Name # NO: Sequence (5′ to 3′) N1    6  4 GTCCCCATCGGAGGGCATCTTATCGTGCCT N2    8  5 CCGCCCTCCTTCGCCCCCCGGTGAAATAAC N3   12  6 AATGCTCACCTCTATTCGGGACTTGAGTAC N4   25  8 GTCGGAACGCCAGGTACAGTTAGCGCATCC N5   27 10 GTGATGCTTTATGAGATTCCGGTCTCCGAC N6   28 11 GACGGTGCATCACCCGCATTTGCTGTAGCG N7   35 13 AAGCCAAAATTACAATCGATCCCTACCAAC N8   37 14 ATCTTGCACCTTCCCAGATGTAAACCCCCT N9   82 21 CGGAGAATACCCTCGACTGTATCATATCGT N10   84 22 TTCATCGAGGTACATTGGTGCTATTCCATT N11   87 24 AGGAGAACCAGCCTGGAGCGTTTAAGCATC N12   88 25 GATGTCCTAAAATGAGGCGTGGCAATAGAG N13   89 26 CAGAGTCATGTATACCCACTGTCGGTCGAA N14  104 27 GTCAGGCTAGGGGGTTATCCCAGCAACGGC N15  116 29 TTTTTGACAGTGATGAAGAGGGAGGTACGA N16  136 31 CTATGGTTCGTTACTGAATCGAAAAGCCGC N17  138 32 TAGCTATCAAAACAGGCGTCATCGGTTAAG N18  152 34 CCTGCTTAGGGTCACTTAAACTACTGGCGC N19  155 35 GGTGATGGCCCATACCGATCACGCCCGCAG N20  156 36 CGGCAGGAGGGACTGCGATTTCCATAGAGC N21  159 37 TGGCCGGAGAGAGGATAGGAAGCGGGACTA N22  163 39 ACACATCCCAGGACTGCCGTGGCCTACGTA N23  174 42 GACGAGCTTGTTCCAATTCCTCGAGCCGAG N24  179 43 GTTGGGGAGGGGCACTACGACTTAGGGCTA N25  185 46 GGCCGTCAATGTGTTTTGCACCCAACCGGA N26  193 48 TCCCACGTCCTTCGACGCACACTGTAACTT N27 >301 49 TCATGTATCGCCCGTGGGTAAGCTC N28 >305 50 ATGTTATGGAGAGTGGGTTAGGCAA N29 >307 51 ATGAGGGAGTAAGGAGATTAGGTTC N30 >313 53 GCCCAGCAGTTATACAATTCGTGGC N31 >314 54 TTGGGCTCTCCAGTAGCCGAACAAA N32 >327 57 GTGGTTGCTACAGCCTAGCCTAGAT N33 >339 59 AGGAGGAACCGGAAGATCTAATCTG N34 >343 61 GCGACTGTGGCAACCCCATTTCGCA N35 >350 63 AGTTGGATGGATATCTCGCTCGTGA N36 >356 64 CGCTGTCCTCTCTGACACTAAAGGT N37 >357 65 ATTTCAATAGTCAACCCGGTATCCA N38 >510 67 GGTTGGGGGGCTCGGCTCATGTATC N39 >517 69 TAAGGAGACTAGGTTCCAATAGCTG N40 >523 70 TTACACAAATCGTGGGTTGGCCTCT N41 >534 71 CGAAAGCGTTCCGCAGGACCCCCTT N42 >541 73 AAAGTCTGAGAATGAGTGATACCAT N43 >544 74 ATATTGGTAGTTTTGTCCGCTGTAG N44 >549 75 CGGAAGATCTAATCTGCACGCAATT N45 >608 76 GCGCCTCGTTGGGCAGAAGTTTGTGGAAAT N46 >610 77 ATCTTCACCTACCGAGTTCTACGGGCCTAC N47 >613 78 CCCACAACTTGCACCCGCTATGCGACCCTG N48 >629 82 GATTAGTGGCCCAACGGGAACAAACTTCCT N49 >701 83 CGCCCGTCCCAGACCCTTACTCACTATGGA N50 >706 85 GAGATTGTACCCTACAGTCCGATTACCGAT N51 >715 86 CGCAGTAAAAGGGCACAGGTAATTACCTTA N52 >718 87 AGGGTGTCTTGAACTACTGGCGCAGCCCAT N53 >723 89 TCGGCGGCGGGTAGTCAGTTCGCTACCTGG N54 >751 94 GATGTGATCTGGACCCTACGGGAGGGGACA N55 >757 96 AGTCCCAGATATGAGAGAAGCGAAGCATAA N56 >815 98 ACTATTACACCACGTACCGTAGGTC

The above sequences were synthesized either as the tag alone (as shown in Table 2 above) or as an oligonucleotide containing both a target-specific sequence and a tag sequence (TS-tag) and were tested as described in the following paragraphs:

Prostate cancer markers PCA3, PSA, T2:ERGa and CAP were used as target nucleic acids in these examples. However, the target nucleic acids that can be used in the presently claimed methods are not hereby limited.

A TMA nucleic acid assay with PCA3 as a target nucleic acid was used to evaluate the candidate tag sequences. Testing was performed with 4 replicates of 2 different PCA3 concentration levels. Results were compared with those obtained using a tag which has been characterized in other amplification assays, the NT7 tag, also referred to as “U20.” All assays used the same T7 provider.

Some representative real time curves from the NT7 tag evaluation are shown in FIGS. 4-6. Performance of the different tags varied in this assay. Some performed approximately the same as U20, some performed much more poorly and some performed better. The best performing previously uncharacterized tags are N47, N48 and N49, which yielded dramatic decreases in emergence times. These results demonstrate the power of this technique in identifying tags with preferable characteristics.

Example 2: Screen for T7 Tag Sequences

A similar strategy to Example 1 was performed for screening tags for use with the T7 provider. A total of 22 sequences were identified for experimental testing and shown in Table 3.

TABLE 3 Sequences Selected for Screening of T7 Tags Sequence Tag SEQ ID Sequence # Name NO: (5′ to 3′) >1003 T1 103 5′ TGGCTAATCCCG >1005 T2 105 5′ CTGTGCTAGAGG >1006 T3 106 5′ CATGTACCAACG >1007 T4 108 5′ TCGGTCGGACTA >1011 T5 109 5′ CCTCCCCCAAGC >1012 T6 110 5′ GGGTTTGCTACG >1014 T7 111 5′ ATGTGCGCACAA >1022 T8 112 5′ CGGGACTAGAGA >1026 T9 113 5′ AATCTCCGAGCG >1034 T10 114 5′ AAGTGCAGGTTC >1042 T11 115 5′ TCCAGTTTAACC >1043 T12 116 5′ TAGCCGCACAGG >1003b T13 104 5′ GCGTTGGCTAAT CCCG >1006b T14 107 5′ TCACCATGTACC AACG >1054 T15 117 5′ TATGAATGCGAC CCGGAA >1063 T16 118 5′ AACAATGGTCAC TGCATC >1066 T17 119 5′ GGGCCGTTTCCC GGACATAA >1067 T18 120 5′ AGGTTGAGTCCG CATCTGAA >1070 T19 121 5′ TCGACCAAGAGC CGCTAGATGC >1076 T20 122 5′ AGCTCGTGTCAA GCCGTCGCCT >1083 T21 123 5′ TGAAAGAGTTGT CAGTTTGCTGGT >1084 T22 124 5′ TCAGGTAAAGGT TCCTCACGCTACC

The sequences of Table 3 were synthesized either as the tag alone (as shown in the table) plus a T7 promoter sequence [5′-aatttaatacgactcactatagggaga-3] or as an oligonucleotide containing both a target-specific sequence and a tag sequence plus the T7 promoter sequence (i.e., construct of TS-tag=T7) and tested as described in the following paragraphs:

The-TMA assay (which in this case used a directly hybridized amplification oligomer complex with the “cPRO” configuration (US Published Application 2008-0305482)) with PCA3 as a target was used in the initial evaluation of the candidate tag sequences. That is, evaluated in a uniplex amplification reaction. Testing was performed with 4 replicates of 2 different PCA3 target levels. Results were compared with a previously characterized tag incorporated into a T7 promoter-based amplification oligomer, referred to as “12 in [5′-CCACAACGGTTT-3]” All assays in this initial evaluation used the same NT7 primer comprising a previously characterized tag (U20).

Some representative real time curves from the T7 tag evaluation are shown in FIGS. 7-9. Performance of the different tags varied in this assay. Some performed approximately the same as the standard 12 in, some performed much more poorly and some performed better. The best performing previously uncharacterized tags in this system were T9, T14, T15, T16, T17, T20, T21 and T22, which yielded dramatic decreases in emergence times. One of the best performing tags—T15—was further tested using PSA as a target nucleic acid, the results of which are shown in FIG. 10. Again, performance was dramatically improved using tag sequences identified by the current methods. These results demonstrate the power of this technique in identifying tags with preferable characteristics.

Example 3: Testing Combinations of NT7 and T7 Tags

Combinations of NT7 and T7 tags are also tested using different TMA formats (see FIGS. 11-17). Combinations are identified whose performance is superior to that of the standard U20/12 in pair, again demonstrating the power of this technique in identifying tags with preferable characteristics.

Example 4: Further Refining Tag Sequences

As mentioned above, once good tag sequences have been identified, performance can be further optimized by the process of incremental changes and subsequent screening. An example of this is depicted in FIGS. 18-19, wherein the T7 tag T21 is systematically shortened and then tested in a TMA amplification assay. The T21 tag sequence was shortened by removing residues from the 3′ end of the tag sequence. Shortening the T21 tag sequence by 2 nucleotides (“T21-2”) slightly improved performance. Shortening the T21 tag sequence by a total of 6 or 8 nucleotides dramatically decreased its performance in this assay. Unexpectedly, shortening the T21 tag sequence even further by removing a total of 10 or 12 nucleotides restored some (although not all) of the original activity (especially 10 nucleotides shorter). This demonstrates the ability of the method to identify sequences possessing unexpected activities, such as the example here wherein a very short tag yielded good performance. Further refining the tag sequence, as illustrated here, is useful for “tuning” the tagged oligonucleotide to perform at a certain level. In some instances, a better performing tagged oligonucleotide is useful; while in other instances decreasing its performance is useful.

It should be noted that the differences in performance identified by this method are useful in a wide variety of ways. For example, the differences in amplification kinetics identified are useful in balancing multiple amplification reactions in a multiplex reaction in order that no one (or more) reactions is so much faster than the others that it (or they) unduly compete for essential shared components in the multiplex amplification mixture. Thus, the method of invention provides a means for evaluating and selecting useful tags by analyzing a variety of performance parameters, by which methods preferred sequence modifications can be identified that would not have been a priori predictable.

Example 5: Screen for NT7 Tag Sequences with Minimal Interference in the PCA3-PSA Duplex Template System Using the Reverse TMA with a Portion of the Amplification Oligomers Containing Tag Sequences

The example described below illustrates the use of the method to select the best tag sequences with minimal interference for use in a universal reverse TMA assay format using the PCA3-PSA target sequence combination. This example is intended only to demonstrate the use of this screening method and does not limit the scope of application to only this assay.

Step 5—Screening in Uniplex:

The 56 NT7 tags that were identified after step 3, and were then synthesized (Table 2) were screened for amplification of a PCA3 target nucleic acid in a uniplex format.

Each tag was screened in a uniplex RUh (reverse half universal TMA, where the universal sequence is on the primer member (NT7) of an amplification oligomer pair comprising a primer and a promoter-based amplification oligomer) reaction for amplifying PCA3 target nucleic acid. Amplification results were evaluated qualitatively. Out of 56 NT7 tags screened, 41 tag sequences optimally amplified PCA3 in this system (see FIG. 20 for representative examples). Separately, 38 NT7 tag sequences were designed and screened for amplification of a PSA target nucleic acid. After qualitative evaluation, 16 of these tags sufficiently amplified PSA (see FIG. 21 for representative examples).

Step 6—Screening Using Duplex Amplification Oligos:

Each of the 41 PCA3 tags passing qualitative criteria in uniplex were then screened in duplex oligo format. PCA3 amplification driven by each tag was observed in presence of PSA oligos containing the U20 standard tag sequence. Similarly, PSA amplification driven by the previously characterized U20 tag was evaluated in the presence of PCA3 amplification oligos with each of 41 tags (see FIGS. 22 and 23 for representative examples). These duplex oligo screens were evaluated qualitatively, and 12 PCA3 tags were confirmed to work well in the presence of PSA oligos comprising standard tag U20.

Each of 16 tag sequences that passed qualitative criteria for PSA amplification in uniplex were then screened against several good tags for PCA3. 27 of these unique tag combinations successfully amplified PCA3 and PSA under duplex oligos conditions.

Step 7—Screening Using Duplex Amplification Oligos and Target Nucleic Acids:

All tag combinations passing qualitative criteria under duplex oligos conditions were subsequently screened in duplex oligos and target sequences format. Amplification of 10³ copies of PCA3 was evaluated in the presence of 10⁶copies of PSA, and 10³copies PSA was evaluated in the presence of 10⁶copies of PCA3. This target input window was chosen based on the interference observed in the standard tag sequence (U20) universal system; specifically, 1000 copies of either PSA or PCA3 target nucleic acid amplified only weakly in the presence of 3-log greater copies of the other target nucleic acid when using a U20 tag (FIG. 24). The levels of template interference with each new tag combination were qualitatively determined relative to the amplification with the U20 standard tag.

Step 8—Interference Analysis:

The interference analysis method was then used to quantitate the magnitude of target sequence interference (I-values) for each tag combination screened in duplex oligos+templates condition. See FIGS. 25 and 26. The tested combinations are shown in FIG. 27 together with the results of the screening test. The lowest I-value of 19.97 for an Analyte Interference Score was seen for the tag sequence combination, Tag N54 (PCA3) and standard tag U20 (PSA). The top 10 tag combinations displaying minimal I-value, are listed in Table 5. A summary of results from all the screens performed for this example is given in FIG. 27.

PCA3 Tag PSA Tag Delta PCA3 Delta PSA Score U20 U20 27 24 51 N54 U20 12 7 19 N54 N209 16 4 20 N34 N201 9 12 21 N14 U20 8 13 21 N54 N226 19 3 22 N54 N216 15 8 23 N14 N216 12 12 24 N34 N216 6 20 25 N14 N207 6 22 28 N14 N201 9 24 33

The best tag combination obtained from this screen was N54/U20. Finally, using the combinations of tags that were selected from the screens, tag N54 (PCA3) and tag U20 (PSA), it was demonstrated that the interference observed was minimal in a Triplex system (PCA3/PSA/IC) compared to the standard system (FIGS. 28 to 31). Reactions conditions in this triplex system were generally set up as follows. For a first set of triplex reactions the non-T7 amplification oligomers for each target nucleic acid (PCA3, PSA and Internal Control) were tagged with the U20 tag sequence (see FIG. 28). Each target capture reaction contained 5 pmol of each target capture oligomer, 5 pmol of each blocker and varied copy numbers of the target nucleic acids according to the chart in FIG. 28 along with internal control. Target capture reactions were performed resulting in capture of the target nucleic acid hybridized with a non-T7 amplification oligomer and a blocker. The captured nucleic acids were then placed into an amplification reaction containing 2.8 pmol, 1.3 pmol and 0.6 pmol of PCA3 T7 amp oligos, PSA T7 amp oligos and internal control T7 amp oligos, respectively; 20 pmol each of PCA3 torch and PSA torch; 10 pmol of internal control torch; and 10 pmol of a non-T7 amplification oligomer targeting the complement of the U20 tag sequence. A real time TMA reaction was performed and the results are shown in FIG. 29. Two nearly identical assays were also performed, the differences being that the non-T7 amplification oligomer targeting PCA3 was tagged with N54 rather than U20. Each of these additional assays contained the same oligomers and target nucleic acids. However, to off-set the impact of a more robust amplification of one of the targets within the multiplex, the T7 concentrations were adjusted. In these additional two reactions, the PCA3 reaction included 3.0-3.8 pmol T7 and 15-20 pmol of torch; the PSA reaction included 15-20 pmol torch and the internal control included 15-20 pmol torch. Other differences compared to the full U20 reaction included using 7.5 or 15 pmol of non-T7 targeting the complement of U20 or N54, respectively. Target copy numbers for these three reactions are shown in FIGS. 28 and 30. Results are shown in FIGS. 29 and 31.

The system using the N54 tag was able to amplify 102 copies of PCA3 and 2491 copies PSA in presence of the highest calibrator level of the competing analyte, whereas the U20 universal “standard” system would not under a balanced set of condition (see FIG. 29). An ‘unbalanced’ system was tested with several different low copy levels in presence of high copy levels of analyte, and the N54/U20/U20 system demonstrated very little interference compared to the U20 standard triplex system (see FIG. 31). Therefore one the advantage of using the tag identification system disclosed herein is that the unbalanced system achieved the same level of quantitation across the dynamic range without interference as did a balanced system.

Thus, the screening method described herein successfully identified tag sequences with minimal multiplex interference in a model system. In general, improved sequence tags provide selective advantage to any under-performing amplification system, and can be selected for a variety of desired properties like speed of amplification, performance, etc, along with minimal interference. In addition, screens can be modified and adapted for identifying improved tags, for alternate TMA formats, other isothermal and non-isothermal amplification formats, including PCR, and in higher plex amplifications. The methods described herein for tag selection can be readily automated to provide a high throughput combinatorial method that quickly screens for tag sequences for several types of assays.

Example 7: Identification and Selection of Tags that are Used in Conjunction with T2:ERGa Target Sequence in T2:ERGa/PSA/IC Triplex TMA Assay System

T2:ERGa is a particularly challenging target nucleic acid for amplification using TMA due, in part, to its Guanine-rich sequence. The standard tag (U20) interferes with T2:ERGa amplification and is not able to amplify and detect the analyte in a universal TMA format. The tags described herein which were identified using the disclosed invention method are able to detect and quantitate T2:ERGa and PSA with sufficient sensitivity, accuracy and precision in the reverse TMA reaction wherein a portion of the amplification oligomers used contain tag sequences.

Previously known tags were not able to amplify T2:ERGa with desired sensitivity and precision. A comparison of the assay performance with the standard tag (U20) and tag N42 is shown in FIG. 32.

The N42 tag, for example, was capable of amplifying T2:ERGa in uniplex as well as in a triplex assay that comprised PSA target sequence and an internal control sequence along with T2:ERGa target nucleic acid. The triplex assay performed by using the N42 tag to amplify all the three target sequences in a one tube multiplex format yielded the desired performance characteristics (see FIGS. 33 and 34).

The N42 tag was also found to be compatible with several other unique tag sequence combinations which are useful in conjunction with T2:ERGa. A couple of examples of assays performed with compatible unique tag sequences in the T2:ERGa/PSA/IC system are shown in FIGS. 35 to 38.

All the tag sequences and combinations disclosed in FIGS. 32-38 are useful for amplification and quantitative detection of T2:ERGa and PSA templates in the T2:ERGa/PSA/IC triplex assay in pure system (using IVT) as well as in clinical urine samples.

TABLE 4 Summary and cross-identification of the sequences described herein and in the Sequence Listing. Oligo SEQ ID Name NO: Sequence 5′ → 3′ Function U20 tag 1 GTCATATGCGACGATCTCAG tag (Std) 1 2 CCCCGTCAAACAAAAACGGGAGCGTGTACC tag 4 3 CCATAGGCCTTCTGCACTGCTCCATATACC tag 6/N1 4 GTCCCCATCGGAGGGCATCTTATCGTGCCT tag 8/N2 5 CCGCCCTCCTTCGCCCCCCGGTGAAATAAC tag 12/N3 6 AATGCTCACCTCTATTCGGGACTTGAGTAC tag 21 7 CCCGCGCACCACCTCCATCACGCAGAAGAG tag 25/N4 8 GTCGGAACGCCAGGTACAGTTAGCGCATCC tag 26 9 AAGTCACTGGCCAGCATAATGCGTGAAGGG tag 27/N5 10 GTGATGCTTTATGAGATTCCGGTCTCCGAC tag 28/N6 11 GACGGTGCATCACCCGCATTTGCTGTAGCG tag 34 12 AGAATTCTTGCAGGTAGAGGTCCCCTCATT tag 35/N7 13 AAGCCAAAATTACAATCGATCCCTACCAAC tag 37/N8 14 ATCTTGCACCTTCCCAGATGTAAACCCCCT tag 42 15 GAAGCGGCAGCTCAGCCGGTTCTCGGAGAG tag 43 16 GCACGCGGGCTCCTTGGGACACTATGATTG tag 61 17 CCCATCAGGACAGTCAGCTGCCCACGAATT tag 78 18 CTTTAGTGCGGTAGGACCGAGACTACCGTG tag 79 19 TTATGTGCCAGCTGGGCCTAAGGCTCCGGG tag 80 20 GACTCTCCTAGGGCGTTCGTCTGGGACTGC tag 82/N9 21 CGGAGAATACCCTCGACTGTATCATATCGT tag 84/N10 22 TTCATCGAGGTACATTGGTGCTATTCCATT tag 86 23 TACCACCTGGTTCAAGGTGTGCCGTACGCG tag 87/N11 24 AGGAGAACCAGCCTGGAGCGTTTAAGCATC tag 88/N12 25 GATGTCCTAAAATGAGGCGTGGCAATAGAG tag 89/N13 26 CAGAGTCATGTATACCCACTGTCGGTCGAA tag 104/N14 27 GTCAGGCTAGGGGGTTATCCCAGCAACGGC tag 106 28 TGGGTTCTGCTAACCGGTGCCGTTCTTAAC tag 116/N15 29 TTTTTGACAGTGATGAAGAGGGAGGTACGA tag 133 30 GAGAACTCGCGCTCCCTCACTCCGTTTAGA tag 136/N16 31 CTATGGTTCGTTACTGAATCGAAAAGCCGC tag 138/N17 32 TAGCTATCAAAACAGGCGTCATCGGTTAAG tag 145 33 AGGACGCTGACACCGTTGGGGTAAAGCGTG tag 152/N18 34 CCTGCTTAGGGTCACTTAAACTACTGGCGC tag 155/N19 35 GGTGATGGCCCATACCGATCACGCCCGCAG tag 156/N20 36 CGGCAGGAGGGACTGCGATTTCCATAGAGC tag 159/N21 37 TGGCCGGAGAGAGGATAGGAAGCGGGACTA tag 161 38 TAGCAGGTGTCTCGGTCCTCAACTGCAAAC tag 163/N22 39 ACACATCCCAGGACTGCCGTGGCCTACGTA tag 171 40 GTGCTAGCCCGGGCCCTTCTTAACTCGGGA tag 172 41 CGGAATCTGAACATCTATCAGAGCCGCGCT tag 174/N23 42 GACGAGCTTGTTCCAATTCCTCGAGCCGAG tag 179/N24 43 GTTGGGGAGGGGCACTACGACTTAGGGCTA tag 182 44 AATGTGGACGGCCGCTCCGTACTTCTGACA tag 183 45 AGGGCCAGCAGCTGGTTCCTTCGCCAGTTA tag 185/N25 46 GGCCGTCAATGTGTTTTGCACCCAACCGGA tag 188 47 CAGTGACTGGGCTAGTGAAGTGAGTCACAG tag 193/N26 48 TCCCACGTCCTTCGACGCACACTGTAACTT tag 301/N27 49 TCATGTATCGCCCGTGGGTAAGCTC tag 305/N28 50 ATGTTATGGAGAGTGGGTTAGGCAA tag 307/N29 51 ATGAGGGAGTAAGGAGATTAGGTTC tag 309 52 CATGCTGCCCGCATACACTTGCGGG tag 313/N30 53 GCCCAGCAGTTATACAATTCGTGGC tag 314/N31 54 TTGGGCTCTCCAGTAGCCGAACAAA tag 316 55 TGACGTTAAACGCAATCCGCGTAAA tag 325 56 GTCGCCATTCAGGACACGCGAAACT tag 327/N32 57 GTGGTTGCTACAGCCTAGCCTAGAT tag 336 58 CCACTTTTCATTCCGAGTCCACGCG tag 339/N33 59 AGGAGGAACCGGAAGATCTAATCTG tag 342 60 CCAATGCTTTCAAATAACCCGTTCT tag 343/N34 61 GCGACTGTGGCAACCCCATTTCGCA tag 346 62 AAAAAACGGAGGAGTCGAACCTTGG tag 350/N35 63 AGTTGGATGGATATCTCGCTCGTGA tag 356/N36 64 CGCTGTCCTCTCTGACACTAAAGGT tag 357/N37 65 ATTTCAATAGTCAACCCGGTATCCA tag 505 66 TTCGCGCCAGCGACCCCACTTATGA tag 510/N38 67 GGTTGGGGGGCTCGGCTCATGTATC tag 516 68 ATGATGCTGAATCGCGATGGGGGGG tag 517/N39 69 TAAGGAGACTAGGTTCCAATAGCTG tag 523/N40 70 TTACACAAATCGTGGGTTGGCCTCT tag 534/N41 71 CGAAAGCGTTCCGCAGGACCCCCTT tag 538 72 CACCCTTGGACACGTGGAAGTGGGC tag 541/N42 73 AAAGTCTGAGAATGAGTGATACCAT tag 544/N43 74 ATATTGGTAGTTTTGTCCGCTGTAG tag 549/N44 75 CGGAAGATCTAATCTGCACGCAATT tag 608/N45 76 GCGCCTCGTTGGGCAGAAGTTTGTGGAAAT tag 610/N46 77 ATCTTCACCTACCGAGTTCTACGGGCCTAC tag 613/N47 78 CCCACAACTTGCACCCGCTATGCGACCCTG tag 618 79 GCCCAGGAGCTCTCCTGGGTAACAGTAGCG tag 620 80 CACGGCCCCCAGGCGGCGTATCAGGGATGA tag 623 81 TCCCCGGCACGGACCGCAAGGGACCAAAGC tag 629/N48 82 GATTAGTGGCCCAACGGGAACAAACTTCCT tag 701/N49 83 CGCCCGTCCCAGACCCTTACTCACTATGGA tag 703 84 GCTACACGCCAGAGGCGCCGCTACAGCGAT tag 706/N50 85 GAGATTGTACCCTACAGTCCGATTACCGAT tag 715/N51 86 CGCAGTAAAAGGGCACAGGTAATTACCTTA tag 718/N52 87 AGGGTGTCTTGAACTACTGGCGCAGCCCAT tag 721 88 CCGCAATCCGGTGACGGCCGGACCGGCAGG tag 723/N53 89 TCGGCGGCGGGTAGTCAGTTCGCTACCTGG tag 729 90 CCAGGACTGCCGTGGCCCACGCACTCACGA tag 740 91 TTGACGCAGGCCCCCGGGGCGACTTCATAC tag 743 92 CGAAAGGAGTTCGAGTGTATCCGGAAGGCG tag 745 93 AGGCGCACTGCGACTTAGGGCTAGCCCCCC tag 751/N54 94 GATGTGATCTGGACCCTACGGGAGGGGACA tag 754 95 TGGGCTGGGGGAGTGAGTCGCTCCCGCAGC tag 757/N55 96 AGTCCCAGATATGAGAGAAGCGAAGCATAA tag 805 97 CGTTTCAGCATCGATGTCCTAAAAT tag 815/N56 98 ACTATTACACCACGTACCGTAGGTC tag 816 99 GGGCAACACCGCGAGCTAATTATCC tag 818 100 GCGCGCGGCCGAGAATCGTTGGAGG tag 826 101 CGCGTCGGGCTTTCGTCTACCCTGG tag 829 102 GGGCGGCCACCGGGGGACCCTGCCC tag 1003/T1 103 TGGCTAATCCCG tag 1003b/T13 104 GCGTTGGCTAATCCCG tag 1005/T2 105 CTGTGCTAGAGG tag 1006/T3 106 CATGTACCAACG tag 1006b/T14 107 TCACCATGTACCAACG tag 1007/T4 108 TCGGTCGGACTA tag 1011/T5 109 CCTCCCCCAAGC tag 1012/T6 110 GGGTTTGCTACG tag 1014/T7 111 ATGTGCGCACAA tag 1022/T8 112 CGGGACTAGAGA tag 1026/T9 113 AATCTCCGAGCG tag 1034/T10 114 AAGTGCAGGTTC tag 1042/T11 115 TCCAGTTTAACC tag 1043/T12 116 TAGCCGCACAGG tag 1054/T15 117 TATGAATGCGACCCGGAA tag 1063/T16 118 AACAATGGTCACTGCATC tag 1066/T17 119 GGGCCGTTTCCCGGACATAA tag 1067/T18 120 AGGTTGAGTCCGCATCTGAA tag 1070/T19 121 TCGACCAAGAGCCGCTAGATGC tag 1076/T20 122 AGCTCGTGTCAAGCCGTCGCCT tag 1083/T21 123 TGAAAGAGTTGTCAGTTTGCTGGT tag 1084/T22 124 TCAGGTAAAGGTTCCTCACGCTACC tag N200 125 CCCATAACTTGGTGCGAATACGGGT tag N201 126 CGTAGCAATGTTCGTCTGACTATGA tag N202 127 CAACTACGGGGATTCTTGGAGAGCC tag N203 128 GTGTAGTATTAGCAAACGATAAGTC tag N204 129 CGGGGGCTGGGAATCTGTGACATGA tag N205 130 TGCCTGTCGATCCATAGGACTCGTG tag N206 131 GAAATGTCCGGGGCCAAAGACAACC tag N207 132 CTGACATAGTATAGCATAGATATTG tag N208 133 GAATTTATAGATACTGCCAATCTAG tag N209 134 ATCAGTTGGACAGAGGGCTGTGTTA tag N210 135 CTTCTAGAGAAGAAGAGTACTGACT tag N211 136 GGTTCAGTTGTAACCATATACTTAC tag N212 137 AATGACGTAGCTATGTATTTTGCAC tag N213 138 AGGTAGCCAACGGGTTTCACATTTC tag N214 139 GCGTAAACTACGATGGCACCTACTC tag N215 140 CTCATAACTTGGTGCGAATACGGGT tag N216 141 TGTAGCAATGTTCGTCTGACTATGA tag N217 142 TAAAATAGTACAGCTACTGGTGATC tag N218 143 CAACTACGAGGATTTTTAGAGAGCC tag N219 144 ATGTAGTATTAGCAAACAATAAGTC tag N220 145 AATTGAATGGAGTCTGATCAATCTT tag N221 146 GAAGTTGGAGGATTAACGTGGGAAT tag N222 147 GGTTTACTATTGTCTCTAATGGGAG tag N223 148 TTGACATAGTATAGCATAGATATTG tag N224 149 CAGATAACTTACCTACATTGAAAGT tag N225 150 TATAGACGACTATTCCGACTAGCAA tag N226 151 GAATTTATAGATACTACCAATCTAG tag N227 152 ATTAATTGGACAGAGGGCTGTGTTA tag N228 153 CTGTTGCCACTCTTTAGAAAGATTA tag N301 154 ACTACAATAATACCAACTATTTGCC tag N302 155 GATACTAAATAACAACTTAGTTTTT tag N303 156 TAGATTTCATTCCGAGTCCACATGT tag N304 157 AACTCTAATATAAGATATCAAGTTA tag N305 158 ATTGTTAAAGTAGACTAATTATCTA tag N306 159 GAAGGAACTGGAAGATTTAATTTGC tag N307 160 ACGCAATTAATATACATATTTATAC tag N308 161 CAATTATGCGAATTCCATTTCACAT tag N309 162 CTTATGAGATGTTAGATATAGTATT tag N310 163 CTTTTACAATATCAGACTTTAGCAA tag N311 164 GATGTAGACGGATTCCATAGAATTT tag N312 165 AATGATTGTGTGGAGTACAAACCAA tag N313 166 TTTTTTTGGCGTAAAGTCTAGAGTT tag N314 167 ATCACGTAAGACCACTGTTAGTATA tag N315 168 CTTTAATAGTCAAACCGATATCCAT tag N316 169 CAGTCAAGTGATGGACTCTAACACA tag N317 170 TCATTAGCGGAAAAAACTGACCTTC tag N318 171 CTCCTATCCTTCGCCACAACTTTAG tag N319 172 TTGCTTTGAGATTGAAATATAAAAG tag N320 173 ATCATATACAGTGCCAGGGAACAAC tag N321 174 ACTTGTAGAAATACCTTATAAAGTT tag N322 175 ATTCTTGATGTATGTAGAGTCCTAA tag N323 176 TGATATCGAATACATAAGTACTCGA tag N324 177 ATGACTGAATTGCTTACACATTTAA tag N325 178 AAAAACAATTAGTATATAACTATTA tag N326 179 TAATAGTGTCATCGGCTCCACTTAT tag N327 180 TACAATCAAAACGTGAAGTTATTGA tag N328 181 GTTTAGTTATTGACTTGTAGATAGT tag N329 182 CTCGACACCGAGTGCTAGATCAACG tag N330 183 ACCCGGACATATTGGCTATTCAAAC tag N331 184 AATATTTAAAAGCCTGGTTTATGTA tag N332 185 CTTTAGTGCCGATTTACGGCCTTGG tag N333 186 GGTAAGATAACGAAGTTTTAATAGC tag N334 187 TGCTTTGACACTGTTCATTATACCG tag N335 188 TTTTCTTTTACCCACTGGTGAAATA tag N336 189 TCAAGATTGTCCTTGATTGTTGAAT tag N337 190 AAAGATCTGATTAACTTATAACAGA tag N338 191 ATGAATAAATCTTGTAAAGTGTGGC tag N339 192 AGCTACACTAAACCTAGAATGATCT tag N340 193 CTTCAATTTGAGACTTGAAATCTAA tag N341 194 GTTTCACTCAGTGTAGACATCATCC tag N342 195 TGGTATCTGAATTACTGCTTTGTCA tag N343 196 AAGTGTCTATTATCCTTAAACGCAT tag N344 197 ATCTCGCATAATAACTCCTCAATAT tag N345 198 GAGTTAGTCTTGTGCTCACGGAATT tag N346 199 AAATGTTAGTTAGCTCGTTCAAGTA tag N347 200 AAAGTTCTTCACACTACGTCAAAAT tag N348 201 AAAAAAATGGTTGTAACAAAAAAAA tag 202 GGCTCATCGATGACCCAAGATGGCGGC Promoter 203 TCTCCCTATAGTGAGTCGTATTAAATT Reverse Complement Promoter 204 GTCTAAGTAGTGACATGTTT PCA3 target specific portion for promoter primer 205 TGGCTAATCCCGGTCTAAGTAGTGACATGTTT T1/PCA3 T1 206 GGCTCATCGATGACCCAAGATGGCGGCTGGCTAATCCCG Prom/T1 T1b 207 GGCTCATCGATGACCCAAGATGGCGGCTGGCTAATCCCGGT Prom/T1/PCA3 CTAAGTAGTGACATGTTT 208 CTGTGCTAGAGGGTCTAAGTAGTGACATGTTT T2/PCA3 T2 209 GGCTCATCGATGACCCAAGATGGCGGCCTGTGCTAGAGG Prom/T2 T2b 210 GGCTCATCGATGACCCAAGATGGCGGCCTGTGCTAGAGGGT Prom/T2/PCA3 CTAAGTAGTGACATGTTT 211 CATGTACCAACGGTCTAAGTAGTGACATGTTT T3/PCA3 T3 212 GGCTCATCGATGACCCAAGATGGCGGCCATGTACCAACG Prom/T3 T3b 213 GGCTCATCGATGACCCAAGATGGCGGCCATGTACCAACGGT Prom/T3/PCA3 CTAAGTAGTGACATGTTT 214 TCGGTCGGACTAGTCTAAGTAGTGACATGTTT T4/PCA3 T4 215 GGCTCATCGATGACCCAAGATGGCGGCTCGGTCGGACTA Prom/T4 T4b 216 GGCTCATCGATGACCCAAGATGGCGGCTCGGTCGGACTAGT Prom/T4/PCA3 CTAAGTAGTGACATGTTT 217 CCTCCCCCAAGCGTCTAAGTAGTGACATGTTT T5/PCA3 T5 218 GGCTCATCGATGACCCAAGATGGCGGCCCTCCCCCAAGC Prom/T5 T5b 219 GGCTCATCGATGACCCAAGATGGCGGCCCTCCCCCAAGCGT Prom/T5/PCA3 CTAAGTAGTGACATGTTT 220 GGGTTTGCTACGGTCTAAGTAGTGACATGTTT T6/PCA3 T6 221 GGCTCATCGATGACCCAAGATGGCGGCGGGTTTGCTACG Prom/T6 T6b 222 GGCTCATCGATGACCCAAGATGGCGGCGGGTTTGCTACGGT Prom/T6/PCA3 CTAAGTAGTGACATGTTT 223 ATGTGCGCACAAGTCTAAGTAGTGACATGTTT T7/PCA3 T7 224 GGCTCATCGATGACCCAAGATGGCGGCATGTGCGCACAA Prom/T7 T7b 225 GGCTCATCGATGACCCAAGATGGCGGCATGTGCGCACAAGT Prom/T7/PCA3 CTAAGTAGTGACATGTTT 226 CGGGACTAGAGAGTCTAAGTAGTGACATGTTT T8/PCA3 T8 227 GGCTCATCGATGACCCAAGATGGCGGCCGGGACTAGAGA Prom/T8 T8b 228 GGCTCATCGATGACCCAAGATGGCGGCCGGGACTAGAGAGT Prom/T8/PCA3 CTAAGTAGTGACATGTTT 229 AATCTCCGAGCGGTCTAAGTAGTGACATGTTT T9/PCA3 T9 230 GGCTCATCGATGACCCAAGATGGCGGCAATCTCCGAGCG Prom/T9 T9b 231 GGCTCATCGATGACCCAAGATGGCGGCAATCTCCGAGCGGT Prom/T9/PCA3 CTAAGTAGTGACATGTTT 232 AAGTGCAGGTTCGTCTAAGTAGTGACATGTTT T10/PCA3 T10 233 GGCTCATCGATGACCCAAGATGGCGGCAAGTGCAGGTTC Prom/T10 T10b 234 GGCTCATCGATGACCCAAGATGGCGGCAAGTGCAGGTTCGT Prom/T10/PCA3 CTAAGTAGTGACATGTTT 235 TCCAGTTTAACCGTCTAAGTAGTGACATGTTT T11/PCA3 T11 236 GGCTCATCGATGACCCAAGATGGCGGCTCCAGTTTAACC Prom/T11 T11b 237 GGCTCATCGATGACCCAAGATGGCGGCTCCAGTTTAACCGT Prom/T11/PCA3 CTAAGTAGTGACATGTTT 238 TAGCCGCACAGGGTCTAAGTAGTGACATGTTT T12/PCA3 T12 239 GGCTCATCGATGACCCAAGATGGCGGCTAGCCGCACAGG Prom/T12 T12b 240 GGCTCATCGATGACCCAAGATGGCGGCTAGCCGCACAGGGT Prom/T12/PCA3 CTAAGTAGTGACATGTTT 241 GCGTTGGCTAATCCCGGTCTAAGTAGTGACATGTTT T13/PCA3 T13 242 GGCTCATCGATGACCCAAGATGGCGGCGCGTTGGCTAATCC Prom/T13 CG T13b 243 GGCTCATCGATGACCCAAGATGGCGGCGCGTTGGCTAATCC Prom/T13/PCA3 CGGTCTAAGTAGTGACATGTTT 244 TCACCATGTACCAACGGTCTAAGTAGTGACATGTTT T14/PCA3 T14 245 GGCTCATCGATGACCCAAGATGGCGGCTCACCATGTACCAA Prom/T14 CG T14b 246 GGCTCATCGATGACCCAAGATGGCGGCTCACCATGTACCAA Prom/T14/PCA3 CGGTCTAAGTAGTGACATGTTT 247 TATGAATGCGACCCGGAAGTCTAAGTAGTGACATGTTT T15/PCA3 T15 248 GGCTCATCGATGACCCAAGATGGCGGCTATGAATGCGACCC Prom/T15 GGAA T15b 249 GGCTCATCGATGACCCAAGATGGCGGCTATGAATGCGACCC Prom/T15/PCA3 GGAAGTCTAAGTAGTGACATGTTT 250 AACAATGGTCACTGCATCGTCTAAGTAGTGACATGTTT T16/PCA3 T16 251 GGCTCATCGATGACCCAAGATGGCGGCAACAATGGTCACTG Prom/T16 CATC T16b 252 GGCTCATCGATGACCCAAGATGGCGGCAACAATGGTCACTG Prom/T16/PCA3 CATCGTCTAAGTAGTGACATGTTT 253 GGGCCGTTTCCCGGACATAAGTCTAAGTAGTGACATGTTT T17/PCA3 T17 254 GGCTCATCGATGACCCAAGATGGCGGCGGGCCGTTTCCCGG Prom/T17 ACATAA T17b 255 GGCTCATCGATGACCCAAGATGGCGGCGGGCCGTTTCCCGG Prom/T17/PCA3 ACATAAGTCTAAGTAGTGACATGTTT 256 AGGTTGAGTCCGCATCTGAAGTCTAAGTAGTGACATGTTT T18/PCA3 T18 257 GGCTCATCGATGACCCAAGATGGCGGCAGGTTGAGTCCGCA Prom/T18 TCTGAA T18b 258 GGCTCATCGATGACCCAAGATGGCGGCAGGTTGAGTCCGCA Prom/T18/PCA3 TCTGAAGTCTAAGTAGTGACATGTTT 259 TCGACCAAGAGCCGCTAGATGCGTCTAAGTAGTGACATGTT T19/PCA3 T T19 260 GGCTCATCGATGACCCAAGATGGCGGCTCGACCAAGAGCCG Prom/T19 CTAGATGC T19b 261 GGCTCATCGATGACCCAAGATGGCGGCTCGACCAAGAGCCG Prom/T19/PCA3 CTAGATGCGTCTAAGTAGTGACATGTTT 262 AGCTCGTGTCAAGCCGTCGCCTGTCTAAGTAGTGACATGTT T20/PCA3 T T20 263 GGCTCATCGATGACCCAAGATGGCGGCAGCTCGTGTCAAGC Prom/T20 CGTCGCCT T20b 264 GGCTCATCGATGACCCAAGATGGCGGCAGCTCGTGTCAAGC Prom/T20/PCA3 CGTCGCCTGTCTAAGTAGTGACATGTTT 265 TGAAAGAGTTGTCAGTTTGCTGGTGTCTAAGTAGTGACATG T21/PCA3 TTT T21 266 GGCTCATCGATGACCCAAGATGGCGGCTGAAAGAGTTGTCA Prom/T21 GTTTGCTGGT T21b 267 GGCTCATCGATGACCCAAGATGGCGGCTGAAAGAGTTGTCA Prom/T21/PCA3 GTTTGCTGGTGTCTAAGTAGTGACATGTTT 268 TCAGGTAAAGGTTCCTCACGCTACCGTCTAAGTAGTGACAT T22/PCA3 GTTT T22 269 GGCTCATCGATGACCCAAGATGGCGGCTCAGGTAAAGGTTC Prom/T22 CTCACGCTACC T22b 270 GGCTCATCGATGACCCAAGATGGCGGCTCAGGTAAAGGTTC Prom/T22/PCA3 CTCACGCTACCGTCTAAGTAGTGACATGTTT 271 GGCTCATCGATGACCCAAGATGGCGGC PCA3 target specific sequence for non- promoter primer RPCA3 21 272 GTCATATGCGACGATCTCAGGGCTCATCGATGACCCAAGAT U20/PCA3 U20 GGCGGC N1b 273 GTCCCCATCGGAGGGCATCTTATCGTGCCTGGCTCATCGAT N1/PCA3 GACCCAAGATGGCGGC non-prom N2b 274 CCGCCCTCCTTCGCCCCCCGGTGAAATAACGGCTCATCGAT N2/PCA3 GACCCAAGATGGCGGC non-prom N3b 275 AATGCTCACCTCTATTCGGGACTTGAGTACGGCTCATCGAT N3/PCA3 GACCCAAGATGGCGGC non-prom N4b 276 GTCGGAACGCCAGGTACAGTTAGCGCATCCGGCTCATCGAT N4/PCA3 GACCCAAGATGGCGGC non-prom N5b 277 GTGATGCTTTATGAGATTCCGGTCTCCGACGGCTCATCGAT N5/PCA3 GACCCAAGATGGCGGC non-prom N6b 278 GACGGTGCATCACCCGCATTTGCTGTAGCGGGCTCATCGAT N6/PCA3 GACCCAAGATGGCGGC non-prom N7b 279 AAGCCAAAATTACAATCGATCCCTACCAACGGCTCATCGAT N7/PCA3 GACCCAAGATGGCGGC non-prom N8b 280 ATCTTGCACCTTCCCAGATGTAAACCCCCTGGCTCATCGAT N8/PCA3 GACCCAAGATGGCGGC non-prom N9b 281 CGGAGAATACCCTCGACTGTATCATATCGTGGCTCATCGAT N9/PCA3 GACCCAAGATGGCGGC non-prom N10b 282 TTCATCGAGGTACATTGGTGCTATTCCATTGGCTCATCGAT N10/PCA3 GACCCAAGATGGCGGC non-prom N11b 283 AGGAGAACCAGCCTGGAGCGTTTAAGCATCGGCTCATCGAT N11/PCA3 GACCCAAGATGGCGGC non-prom N12b 284 GATGTCCTAAAATGAGGCGTGGCAATAGAGGGCTCATCGAT N12/PCA3 GACCCAAGATGGCGGC non-prom N13b 285 CAGAGTCATGTATACCCACTGTCGGTCGAAGGCTCATCGAT N13/PCA3 GACCCAAGATGGCGGC non-prom N14b 286 GTCAGGCTAGGGGGTTATCCCAGCAACGGCGGCTCATCGAT N14/PCA3 GACCCAAGATGGCGGC non-prom N15b 287 TTTTTGACAGTGATGAAGAGGGAGGTACGAGGCTCATCGAT N15/PCA3 GACCCAAGATGGCGGC non-prom N16b 288 CTATGGTTCGTTACTGAATCGAAAAGCCGCGGCTCATCGAT N16/PCA3 GACCCAAGATGGCGGC non-prom N17b 289 TAGCTATCAAAACAGGCGTCATCGGTTAAGGGCTCATCGAT N17/PCA3 GACCCAAGATGGCGGC non-prom N18b 290 CCTGCTTAGGGTCACTTAAACTACTGGCGCGGCTCATCGAT N18/PCA3 GACCCAAGATGGCGGC non-prom N19b 291 GGTGATGGCCCATACCGATCACGCCCGCAGGGCTCATCGAT N19/PCA3 GACCCAAGATGGCGGC non-prom N20b 292 CGGCAGGAGGGACTGCGATTTCCATAGAGCGGCTCATCGAT N20/PCA3 GACCCAAGATGGCGGC non-prom N21b 293 TGGCCGGAGAGAGGATAGGAAGCGGGACTAGGCTCATCGAT N21/PCA3 GACCCAAGATGGCGGC non-prom N22b 294 ACACATCCCAGGACTGCCGTGGCCTACGTAGGCTCATCGAT N22/PCA3 GACCCAAGATGGCGGC non-prom N23b 295 GACGAGCTTGTTCCAATTCCTCGAGCCGAGGGCTCATCGAT N23/PCA3 GACCCAAGATGGCGGC non-prom N24b 296 GTTGGGGAGGGGCACTACGACTTAGGGCTAGGCTCATCGAT N24/PCA3 GACCCAAGATGGCGGC non-prom N25b 297 GGCCGTCAATGTGTTTTGCACCCAACCGGAGGCTCATCGAT N25/PCA3 GACCCAAGATGGCGGC non-prom N26b 298 TCCCACGTCCTTCGACGCACACTGTAACTTGGCTCATCGAT N26/PCA3 GACCCAAGATGGCGGC non-prom N27b 299 TCATGTATCGCCCGTGGGTAAGCTCGGCTCATCGATGACCC N27/PCA3 AAGATGGCGGC non-prom N28b 300 ATGTTATGGAGAGTGGGTTAGGCAAGGCTCATCGATGACCC N28/PCA3 AAGATGGCGGC non-prom N29b 301 ATGAGGGAGTAAGGAGATTAGGTTCGGCTCATCGATGACCC N29/PCA3 AAGATGGCGGC non-prom N30b 302 GCCCAGCAGTTATACAATTCGTGGCGGCTCATCGATGACCC N30/PCA3 AAGATGGCGGC non-prom N31b 303 TTGGGCTCTCCAGTAGCCGAACAAAGGCTCATCGATGACCC N31/PCA3 AAGATGGCGGC non-prom N32b 304 GTGGTTGCTACAGCCTAGCCTAGATGGCTCATCGATGACCC N32/PCA3 AAGATGGCGGC non-prom N33b 305 AGGAGGAACCGGAAGATCTAATCTGGGCTCATCGATGACCC N33/PCA3 AAGATGGCGGC non-prom N34b 306 GCGACTGTGGCAACCCCATTTCGCAGGCTCATCGATGACCC N34/PCA3 AAGATGGCGGC non-prom N35b 307 AGTTGGATGGATATCTCGCTCGTGAGGCTCATCGATGACCC N35/PCA3 AAGATGGCGGC non-prom N36b 308 CGCTGTCCTCTCTGACACTAAAGGTGGCTCATCGATGACCC N36/PCA3 AAGATGGCGGC non-prom N37b 309 ATTTCAATAGTCAACCCGGTATCCAGGCTCATCGATGACCC N37/PCA3 AAGATGGCGGC non-prom N38b 310 GGTTGGGGGGCTCGGCTCATGTATCGGCTCATCGATGACCC N38/PCA3 AAGATGGCGGC non-prom N39b 311 TAAGGAGACTAGGTTCCAATAGCTGGGCTCATCGATGACCC N39/PCA3 AAGATGGCGGC non-prom N40b 312 TTACACAAATCGTGGGTTGGCCTCTGGCTCATCGATGACCC N40/PCA3 AAGATGGCGGC non-prom N41b 313 CGAAAGCGTTCCGCAGGACCCCCTTGGCTCATCGATGACCC N41/PCA3 AAGATGGCGGC non-prom N42b 314 AAAGTCTGAGAATGAGTGATACCATGGCTCATCGATGACCC N42/PCA3 AAGATGGCGGC non-prom N43b 315 ATATTGGTAGTTTTGTCCGCTGTAGGGCTCATCGATGACCC N43/PCA3 AAGATGGCGGC non-prom N44b 316 CGGAAGATCTAATCTGCACGCAATTGGCTCATCGATGACCC N44/PCA3 AAGATGGCGGC non-prom N45b 317 GCGCCTCGTTGGGCAGAAGTTTGTGGAAATGGCTCATCGAT N45/PCA3 GACCCAAGATGGCGGC non-prom N46b 318 ATCTTCACCTACCGAGTTCTACGGGCCTACGGCTCATCGAT N45/PCA3 GACCCAAGATGGCGGC non-prom N47b 319 CCCACAACTTGCACCCGCTATGCGACCCTGGGCTCATCGAT N47/PCA3 GACCCAAGATGGCGGC non-prom N48b 320 GATTAGTGGCCCAACGGGAACAAACTTCCTGGCTCATCGAT N48/PCA3 GACCCAAGATGGCGGC non-prom N49b 321 CGCCCGTCCCAGACCCTTACTCACTATGGAGGCTCATCGAT N49/PCA3 GACCCAAGATGGCGGC non-prom N50b 322 GAGATTGTACCCTACAGTCCGATTACCGATGGCTCATCGAT N50/PCA3 GACCCAAGATGGCGGC non-prom N51b 323 CGCAGTAAAAGGGCACAGGTAATTACCTTAGGCTCATCGAT N51/PCA3 GACCCAAGATGGCGGC non-prom N52b 324 AGGGTGTCTTGAACTACTGGCGCAGCCCATGGCTCATCGAT N52/PCA3 GACCCAAGATGGCGGC non-prom N53b 325 TCGGCGGCGGGTAGTCAGTTCGCTACCTGGGGCTCATCGAT N53/PCA3 GACCCAAGATGGCGGC non-prom N54b 326 GATGTGATCTGGACCCTACGGGAGGGGACAGGCTCATCGAT N54/PCA3 GACCCAAGATGGCGGC non-prom N55b 327 AGTCCCAGATATGAGAGAAGCGAAGCATAAGGCTCATCGAT N55/PCA3 GACCCAAGATGGCGGC non-prom N56b 328 ACTATTACACCACGTACCGTAGGTCGGCTCATCGATGACCC N56/PCA3 AAGATGGCGGC non-prom 329 GTCATATGCGACGATCTCAGGGCTCATCGATGACCCAAGAT U20/PCA3 GGCGGC 330 TCTCCCTATAGTGAGTCGTATTAAATTGTCATATGCGACGA RC TCTCAG PROM/U20 PCA3 U20- 331 TCTCCCTATAGTGAGTCGTATTAAATTGTCATATGCGACGA RC cPRO TCTCAGGGCTCATCGATGACCCAAGATGGCGGC Prom/U20/PCA3 (NOTE: PCA3 sequence same as non- promoter primer) 332 GTGATGCTTTATGAGATTCCGGTCTCCGACGGCTCATCGAT N5/PCA3 GACCCAAGATGGCGGC 333 TCTCCCTATAGTGAGTCGTATTAAATTGTGATGCTTTATGA RC PROM/N5 GATTCCGGTCTCCGAC N5b_cPRO 334 TCTCCCTATAGTGAGTCGTATTAAATTGTGATGCTTTATGA RC GATTCCGGTCTCCGACGGCTCATCGATGACCCAAGATGGCG Prom/N5/PCA3 GC 335 TTCATCGAGGTACATTGGTGCTATTCCATTGGCTCATCGAT N10/PCA3 GACCCAAGATGGCGGC 336 TCTCCCTATAGTGAGTCGTATTAAATTTTCATCGAGGTACA RC TTGGTGCTATTCCATT PROM/PCA3 N10_cPRO 337 TCTCCCTATAGTGAGTCGTATTAAATTTTCATCGAGGTACA RC TTGGTGCTATTCCATTGGCTCATCGATGACCCAAGATGGCG Prom/N10/PCA3 GC 338 GATGTCCTAAAATGAGGCGTGGCAATAGAGGGCTCATCGAT N12/PCA3 GACCCAAGATGGCGGC 339 TCTCCCTATAGTGAGTCGTATTAAATTGATGTCCTAAAATG RC AGGCGTGGCAATAGAG PROM/N12 N12_cPRO 340 TCTCCCTATAGTGAGTCGTATTAAATTGATGTCCTAAAATG RC AGGCGTGGCAATAGAGGGCTCATCGATGACCCAAGATGGCG Prom/N12/PCA3 GC 341 CAGAGTCATGTATACCCACTGTCGGTCGAAGGCTCATCGAT N13/PROM GACCCAAGATGGCGGC 342 TCTCCCTATAGTGAGTCGTATTAAATTCAGAGTCATGTATA RC CCCACTGTCGGTCGAA PROM/N13 N13b_cPRO 343 TCTCCCTATAGTGAGTCGTATTAAATTCAGAGTCATGTATA Prom/N13/PCA3 CCCACTGTCGGTCGAAGGCTCATCGATGACCCAAGATGGCG GC 344 ATGTTATGGAGAGTGGGTTAGGCAAGGCTCATCGATGACCC N28/PCA3 AAGATGGCGGC 345 TCTCCCTATAGTGAGTCGTATTAAATTATGTTATGGAGAGT RC PROM GGGTTAGGCAA PCA3 N28b_cPRO 346 TCTCCCTATAGTGAGTCGTATTAAATTATGTTATGGAGAGT RC GGGTTAGGCAAGGCTCATCGATGACCCAAGATGGCGGC Prom/N28/PCA3 347 AAAGTCTGAGAATGAGTGATACCATGGCTCATCGATGACCC N42/PCA3 AAGATGGCGGC 348 TCTCCCTATAGTGAGTCGTATTAAATTAAAGTCTGAGAATG RC AGTGATACCAT RPOM/N42 N42b_cPRO 349 TCTCCCTATAGTGAGTCGTATTAAATTAAAGTCTGAGAATG RC AGTGATACCATGGCTCATCGATGACCCAAGATGGCGGC Prom/N42/PCA3 350 CCCACAACTTGCACCCGCTATGCGACCCTGGGCTCATCGAT N47/PCA3 GACCCAAGATGGCGGC 351 TCTCCCTATAGTGAGTCGTATTAAATTCCCACAACTTGCAC RC CCGCTATGCGACCCTG PROM/N47 N47b_cPRO 352 TCTCCCTATAGTGAGTCGTATTAAATTCCCACAACTTGCAC RC CCGCTATGCGACCCTGGGCTCATCGATGACCCAAGATGGCG Prom/N47/PCA3 GC 353 GATTAGTGGCCCAACGGGAACAAACTTCCTGGCTCATCGAT N48/PCA3 GACCCAAGATGGCGGC 354 TCTCCCTATAGTGAGTCGTATTAAATTGATTAGTGGCCCAA RC CGGGAACAAACTTCCT PROM/N48 N48b_cPRO 355 TCTCCCTATAGTGAGTCGTATTAAATTGATTAGTGGCCCAA RC CGGGAACAAACTTCCTGGCTCATCGATGACCCAAGATGGCG Prom/N48/PCA3 GC 356 CGCCCGTCCCAGACCCTTACTCACTATGGAGGCTCATCGAT N49/PCA3 GACCCAAGATGGCGGC 357 TCTCCCTATAGTGAGTCGTATTAAATTCGCCCGTCCCAGAC RC CCTTACTCACTATGGA PROM/N49 N49b_cPRO 358 TCTCCCTATAGTGAGTCGTATTAAATTCGCCCGTCCCAGAC RC CCTTACTCACTATGGAGGCTCATCGATGACCCAAGATGGCG Prom/N49/PCA3 GC BOmePCA3 359 GAUGCAGUGGGCAGCUGUGAGGAC PCA3 3e3(-) 112- BLOCKER 3′blk RPCA3e3(-) 360 UGUGUCUUCAGGAUGAAACACACA PCA3 PROBE 147-166_C9 (21/22) 361 AUCUGUUUUCCUGCCCAUCCUUUAAG PCA3 TARGET CAPTURE PCA3e4(-) 362 AUCUGUUUUCCUGCCCAUCCUUUAAG TTTAAAAAAAAAAAA PCA3 109dT3A30 AAAAAAAAAAAAAAAA TARGET 3′-blocked CAPTURE 363 CCACTGCATCAGGAACAAAAGCGTGATCTTG PSA target specific sequence for promoter primer 364 TGGCTAATCCCGCCACTGCATCAGGAACAAAAGCGTGATCT T1/PSA TG T1 365 GGCTCATCGATGACCCAAGATGGCGGCTGGCTAATCCCG Prom/T1 PSA T1b 366 GGCTCATCGATGACCCAAGATGGCGGCTGGCTAATCCCGCC Prom/T1/PSA ACTGCATCAGGAACAAAAGCGTGATCTTG 367 AGGTTGAGTCCGCATCTGAACCACTGCATCAGGAACAAAAG T18/PSA CGTGATCTTG T18 368 GGCTCATCGATGACCCAAGATGGCGGCAGGTTGAGTCCGCA Prom/T18 TCTGAA PSA T18b 369 GGCTCATCGATGACCCAAGATGGCGGCAGGTTGAGTCCGCA Prom/T18/PSA TCTGAACCACTGCATCAGGAACAAAAGCGTGATCTTG 370 ATGTGCGCACAACCACTGCATCAGGAACAAAAGCGTGATCT T7/PSA TG T7 371 GGCTCATCGATGACCCAAGATGGCGGCATGTGCGCACAA Prom/T7 PSA T7b 372 GGCTCATCGATGACCCAAGATGGCGGCATGTGCGCACAACC Prom/T7/PSA ACTGCATCAGGAACAAAAGCGTGATCTTG 373 TATGAATGCGACCCGGAACCACTGCATCAGGAACAAAAGCG T15/PSA TGATCTTG T15 374 GGCTCATCGATGACCCAAGATGGCGGCTATGAATGCGACCC Prom/T15 GGAA PSA T15b 375 GGCTCATCGATGACCCAAGATGGCGGCTATGAATGCGACCC Prom/T15/PSA GGAACCACTGCATCAGGAACAAAAGCGTGATCTTG 376 CATGTACCAACGCCACTGCATCAGGAACAAAAGCGTGATCT T3/PSA TG T3 377 GGCTCATCGATGACCCAAGATGGCGGCCATGTACCAACG Prom/T3 PSA T3b 378 GGCTCATCGATGACCCAAGATGGCGGCCATGTACCAACGCC Prom/T3/PSA ACTGCATCAGGAACAAAAGCGTGATCTTG 379 AATCTCCGAGCGCCACTGCATCAGGAACAAAAGCGTGATCT T9/PSA TG T9 380 GGCTCATCGATGACCCAAGATGGCGGCAATCTCCGAGCG Prom/T9 PSA T9b 381 GGCTCATCGATGACCCAAGATGGCGGCAATCTCCGAGCGCC Prom/T9/PSA ACTGCATCAGGAACAAAAGCGTGATCTTG 382 TCACCATGTACCAACGCCACTGCATCAGGAACAAAAGCGTG T14/PSA ATCTTG T14 383 GGCTCATCGATGACCCAAGATGGCGGCTCACCATGTACCAA Prom/T14 CG PSA T14b 384 GGCTCATCGATGACCCAAGATGGCGGCTCACCATGTACCAA Prom/T14/PSA CGCCACTGCATCAGGAACAAAAGCGTGATCTTG 385 AACAATGGTCACTGCATCCCACTGCATCAGGAACAAAAGCG T16/PSA TGATCTTG T16 386 GGCTCATCGATGACCCAAGATGGCGGCAACAATGGTCACTG Prom/T16 CATC PSA T16b 387 GGCTCATCGATGACCCAAGATGGCGGCAACAATGGTCACTG Prom/T16/PSA CATCCCACTGCATCAGGAACAAAAGCGTGATCTTG 388 GGGCCGTTTCCCGGACATAACCACTGCATCAGGAACAAAAG T17/PSA CGTGATCTTG T17 389 GGCTCATCGATGACCCAAGATGGCGGCGGGCCGTTTCCCGG Prom/T17 ACATAA PSA T17b 390 GGCTCATCGATGACCCAAGATGGCGGCGGGCCGTTTCCCGG Prom/T17/PSA ACATAACCACTGCATCAGGAACAAAAGCGTGATCTTG 391 TGAAAGAGTTGTCAGTTTGCTGGTCCACTGCATCAGGAACA T21/PSA AAAGCGTGATCTTG T21 392 GGCTCATCGATGACCCAAGATGGCGGCTGAAAGAGTTGTCA Prom/T21 GTTTGCTGGT PSA T21b 393 GGCTCATCGATGACCCAAGATGGCGGCTGAAAGAGTTGTCA Prom/T21/PSA GTTTGCTGGTCCACTGCATCAGGAACAAAAGCGTGATCTTG 394 TCAGGTAAAGGTTCCTCACGCTACCCCACTGCATCAGGAAC T22/PSA AAAAGCGTGATCTTG T22 395 GGCTCATCGATGACCCAAGATGGCGGCTCAGGTAAAGGTTC Prom/T22 CTCACGCTACC PSA T22b 396 GGCTCATCGATGACCCAAGATGGCGGCTCAGGTAAAGGTTC Prom/T22/PSA CTCACGCTACCCCACTGCATCAGGAACAAAAGCGTGATCTT G 397 GCTGTGGCTGACCTGAAATACC PSA target specific sequence for non- promoter primer PSA N5b 398 GTGATGCTTTATGAGATTCCGGTCTCCGACGCTGTGGCTGA N5/ PSA CCTGAAATACC non-prom PSA N12b 399 GATGTCCTAAAATGAGGCGTGGCAATAGAGGCTGTGGCTGA N12/PSA CCTGAAATACC non-prom PSA N13b 400 CAGAGTCATGTATACCCACTGTCGGTCGAAGCTGTGGCTGA N13/PSA CCTGAAATACC non-prom PSA N28b 401 ATGTTATGGAGAGTGGGTTAGGCAAGCTGTGGCTGACCTGA N28/PSA AATACC non-prom PSA N42b 402 AAAGTCTGAGAATGAGTGATACCATGCTGTGGCTGACCTGA N42/PSA AATACC non-prom PSA N47b 403 CCCACAACTTGCACCCGCTATGCGACCCTGGCTGTGGCTGA N47/PSA CCTGAAATACC non-prom PSA N48b 404 GATTAGTGGCCCAACGGGAACAAACTTCCTGCTGTGGCTGA N48/PSA CCTGAAATACC non-prom PSA N49b 405 CGCCCGTCCCAGACCCTTACTCACTATGGAGCTGTGGCTGA N49/PSA CCTGAAATACC non-prom RPSAe2e3 406 GAUGCAGUGGGCAGCUGUGAGGAC PSA (-)222- BLOCKER 244_BKD RPSAe3(-) 407 UGUGUCUUCAGGAUGAAACACACA PSA PROBE 32-51_C9 (19,20) 408 CGAACUUGCGCACACACGUCAUUGGA PSA TARGET CAPTURE PSA(-) 409 CGAACUUGCGCACACACGUCAUUGGA TTTAAAAAAAAAAAA PSA TARGET 581dT3A30 AAAAAAAAAAAAAAAAAA CAPTURE Key to identity of sequences: Normal- tag sequences Bold- pca3 target specific sequences Double Underline- PSA target specific sequences Underline- promoter sequence Italics- dT(3)A(30) tail

The invention being thus described, it will be apparent to one of ordinary skill in the art that various modifications of the materials and methods for practicing the invention can be made. Such modifications are to be considered within the scope of the invention as defined by the following claims.

Each of the references from the patent and periodical literature cited herein is hereby expressly incorporated in its entirety by such citation. 

1.-44. (canceled)
 45. A nucleic acid tag sequence obtained by a method comprising the steps of: a) generating a pool of nucleic acid sequences, wherein the pool is at least three nucleic acid sequences; b) comparing a nucleic acid sequence or sequences from the pool of nucleic acid sequences against a database having one or more nucleic acid sequences to determine complementarity of the nucleic acid sequences from the pool of nucleic acid sequences to the database having one or more sequences, c) generating a sub-pool of nucleic acid sequences, wherein the sub-pool is a collection of nucleic acid sequences with complementarity that is less than 95% to the nucleic acid sequence(s) in the database, that is less than 90% to the nucleic acid sequence(s) in the database; that is less than 80% to the nucleic acid sequence(s) in the database, that is less than 70% to the nucleic acid sequence(s) in the database, or that is less than 50% to the nucleic acid sequence(s) in the database; d) screening the sub-pool of nucleic acid sequences for one or more performance characteristics selected from melting temperature, activity in an enzyme reaction, G-C content, nucleobase composition, length, hybridization energy, multimer formation, internal structure formation, G-quartet formation, and hairpin-stability, e) selecting one or more nucleic acid sequences from the sub-pool for use as tag sequences in a nucleic acid assay; f) synthesizing at least two different oligonucleotides for use in a nucleic acid assay, wherein each of the synthesized oligonucleotides has a tag sequence selected according to step e); and g) measuring for each of the different oligonucleotides synthesized in step f) one or more of the following performance characteristics: speed of amplification, limit of detection, interference, precision of replicates, performance against a specific target nucleic acid sequence, or performance against multiple target nucleic acid sequences in a nucleic acid assay, and optionally comparing the measurements to the measurements obtained for an untagged oligonucleotide; and h) selecting one or more of the nucleic acid tag sequences used in step g); i) modifying the sequence of the tag sequence incorporated into an oligonucleotide from step h) to obtain a modified tag sequence for incorporation into an oligonucleotide; j) measuring for the oligonucleotide containing a modified tag sequence from step i) one or more of the following performance characteristics: speed of amplification, limit of detection, interference, precision of replicates, performance against a specific target nucleic acid sequence, or performance against multiple target nucleic acid sequences in a nucleic acid assay; and k) selecting one or more of the modified nucleic acid tag sequences used in step j) for use in a nucleic acid assay.
 46. An amplification oligomer having a nucleic acid sequence that includes a tag sequence obtained by a method comprising the steps of: a) generating a pool of nucleic acid sequences, wherein the pool is at least three nucleic acid sequences; b) comparing a nucleic acid sequence or sequences from the pool of nucleic acid sequences against a database having one or more nucleic acid sequences to determine complementarity of the nucleic acid sequences from the pool of nucleic acid sequences to the database having one or more sequences, c) generating a sub-pool of nucleic acid sequences, wherein the sub-pool is a collection of nucleic acid sequences with complementarity that is less than 95% to the nucleic acid sequence(s) in the database, that is less than 90% to the nucleic acid sequence(s) in the database; that is less than 80% to the nucleic acid sequence(s) in the database, that is less than 70% to the nucleic acid sequence(s) in the database, or that is less than 50% to the nucleic acid sequence(s) in the database; d) screening the sub-pool of nucleic acid sequences for one or more performance characteristics selected from melting temperature, activity in an enzyme reaction, G-C content, nucleobase composition, length, hybridization energy, multimer formation, internal structure formation, G-quartet formation, and hairpin-stability, e) selecting one or more nucleic acid sequences from the sub-pool for use as tag sequences in a nucleic acid assay; f) synthesizing at least two different oligonucleotides for use in a nucleic acid assay, wherein each of the synthesized oligonucleotides has a tag sequence selected according to step e); and g) measuring for each of the different oligonucleotides synthesized in step f) one or more of the following performance characteristics: speed of amplification, limit of detection, interference, precision of replicates, performance against a specific target nucleic acid sequence, or performance against multiple target nucleic acid sequences in a nucleic acid assay, and optionally comparing the measurements to the measurements obtained for an untagged oligonucleotide; and h) selecting one or more of the nucleic acid tag sequences used in step g); i) modifying the sequence of the tag sequence incorporated into an oligonucleotide from step h) to obtain a modified tag sequence for incorporation into an oligonucleotide; j) measuring for the oligonucleotide containing a modified tag sequence from step i) one or more of the following performance characteristics: speed of amplification, limit of detection, interference, precision of replicates, performance against a specific target nucleic acid sequence, or performance against multiple target nucleic acid sequences in a nucleic acid assay; and k) selecting one or more of the modified nucleic acid tag sequences used in step j) for use in a nucleic acid assay.
 47. A method for identifying nucleic acid tag sequences for use in an in vitro nucleic acid amplification assay, comprising the steps of: a) generating a pool of nucleic acid sequences, wherein the pool is at least three nucleic acid sequences from Table 1; b) screening the pool of nucleic acid sequences against a database containing one or more nucleic acid sequences to identify percent complementarity between nucleic acid sequences in the pool and nucleic acid sequences in the database; c) screening the pool of nucleic acid sequences to determine a performance characteristic selected from the group consisting of: G-C content, nucleobase composition, length, multimer formation, primer-dimer formation, Tm, hairpin stabilization energy, self dimer stabilization energy, internal structure formation, G-quartet formation, hybridization energy, activity in an enzyme reaction, and combinations thereof; d) generating a sub-pool of nucleic acid sequences from the results obtained in step b), step c) or steps b) and c); e) selecting one or more nucleic acid sequences from the sub-pool for use as tag sequences in a nucleic acid assay; f) synthesizing an amplification oligomer containing a tag sequence selected at step e); and g) performing an in vitro nucleic acid amplification reaction using the amplification oligomer.
 48. (canceled)
 49. The method according to claim 47, wherein the sub-pool at step d) contains one or more of the following: nucleic acid sequences with Tm values that are within ±2 degrees C. from a mean Tm of nucleic acids in the sub-pool; nucleic acid sequences with Tm values that are within ±5 degrees C. from a mean Tm of nucleic acids in the sub-pool; nucleic acid sequences with Tm values that are within ±10 degrees C. from a mean Tm of nucleic acids in the sub-pool; nucleic acid sequences with G-C contents that are within ±5% from the mean G-C content of the nucleic acids in the sub-pool; nucleic acid sequences with G-C contents that are within ±10% from the mean G-C content of the nucleic acids in the sub-pool; nucleic acid sequences with G-C contents that are within ±30% from the mean G-C content of the nucleic acids in the sub-pool; nucleic acid sequences with G-C contents from 30% to 80%, from 40% to 70%, or from 30% to 50%; nucleic acid sequences in Table 2; and nucleic acid sequences with lengths from 5 nucleobases to 100 nucleobases. 50.-57. (canceled)
 58. The method according to claim 47, wherein the in vitro amplification reaction performed at step g) has one or more of the following performance characteristics: reduced interference between nucleic acids in the reaction when performed with the tagged amplification oligomer from step f) compared to when performed using an untagged amplification oligomer; reaction kinetics that are accelerated by about 105% or more when performed with the tagged amplification oligomer from step f) compared to when performed using an untagged amplification oligomer; reaction kinetics that are reduced to about 95% or less when performed with the tagged amplification oligomer from step f) compared to when performed using an untagged amplification oligomer; increased sensitivity when performed with the tagged amplification oligomer from step f) compared to when performed using an untagged amplification oligomer, wherein the in vitro amplification reaction using the tagged amplification oligomer requires an amount of starting material that is about 95% or less than the minimum amount of starting material required in an untagged assay in order to obtain a detectable signal; decreased sensitivity when performed with the tagged amplification oligomer from step f) compared to when performed using an untagged amplification oligomer, wherein the in vitro amplification reaction using the tagged amplification oligomer requires an amount of starting material that is about 105% or more than the amount of starting material required in an untagged assay in order to obtain a detectable signal; and a replication precision that is about 105% or better when performed with the tagged amplification oligomer from step f) compared to when performed using an untagged amplification oligomer. 59.-63. (canceled)
 64. The method according to claim 58, wherein the tagged assay decreases the performance parameter by from 25% to 94%, from 50% to 94%, or from 75% to 94% compared to the untagged assay, wherein each range is inclusive of all whole and partial numbers therein; or wherein the tagged assay increases the performance parameter by from 105% to 150%, from 105% to 200%, or from 105% to 500% compared to the untagged assay, wherein each range is inclusive of all whole and partial numbers therein.
 65. (canceled)
 66. The method according to claim 47, wherein the one or more nucleic acid sequences in a database is a collection of various nucleic acid sequences corresponding to a nucleic acid assay, a public collection of nucleic acid sequences, an aligned collection of nucleic acid sequences, the pool of nucleic acid sequences, or a combination thereof or wherein the one or more nucleic acid sequences in a database contains sequence(s) that are derived from: collections of various nucleic acid sequences corresponding to a nucleic acid assay; a public collection of nucleic acid sequences; a collection of aligned sequences, the pool, or a combination thereof.
 67. (canceled)
 68. The method according to any one of claims 47, 49 58, 64, and 66, wherein the in vitro amplification assay is an isothermal amplification assay; or a multiplex amplification assay; or a PCR amplification reaction; or a combination thereof. 69.-70. (canceled)
 71. The method according to claim 68, wherein an amplicon generated in the in vitro amplification assay is used in a sequencing assay. 72.-109. (canceled) 